Abstract
Purpose :
Difference in the culturing conditions of umbilical cord derived mesenchymal stem cells (UMSC) across different groups causes variation and impacts the therapeutic efficiency of UMSC in treating acquired/congenital ocular surface disorders. We hypothesize that the cells harvested at mid-log phase has better treatment efficacy compared to the cells harvested at early-log phase.
Methods :
UMSCs were cultured at two different densities – 1200 cells/cm2 and 4000 cells/cm2 using serum free media. The cells were harvested and counted at the end of three days. UMSCs on various time-points on the growth curve were analyzed for the expression of transcription factors such as Twist1, Oct4, Nanog and surface marker CD44. Six-week old C57BL/6 mice (n=64) were used for the wound healing experiments. Cells were transplanted using intra stromal injections on alkali burned mouse cornea. Three treatment groups such as treatment with UMSC recovered from liquid nitrogen [UMSC-R] (n=16), UMSC sub cultured and harvested at early log phase [EL UMSC] (n=16) and UMSC harvested at mid-log phase [ML UMSC] (n=16) were transplanted and its therapeutic efficacy was studied. PBS treatment was done for the control group animals. Cornea was imaged every 7 days until day 28 (D28) and percentage of transparent corneas [% TC] was calculated.
Results :
Cells cultured at 1200 cells/cm2 was at early-log phase and 4000 cells/cm2 was at mid-log phase with cell densities of 15000 cells/cm2 and 30,000 cells/cm2 repectively. Flow cytometry analysis suggested that EL UMSCs had higher expression of Twist1, Oct4, Nanog and CD44 whereas ML UMSCs had a lower expression of all the markers. UMSC-R group had increase in % TC from 14% on D7 to 50% on D21 but decreased to 38% on D28. EL UMSC group had increase in % TC from 7% on D7 to 50% on D21 but it decreased to 38% on D28. Transplantation of ML UMSC increased the % TC from 36% on D7 to 63% on D28. Control group animals with PBS had a % TC of 17% on D28. Comparing all groups, the ML UMSC transplantation resulted in higher transparent corneas compared to the EL phase UMSC and UMSC-R.
Conclusions :
Our results suggets that the mid-log phase UMSCs have better therapeutic efficiency compared to early log phase UMSCs. Markers such as Twist1, Oct4, Nanog and CD44 can be considered a good standard for determining the therapeutic efficiency of the UMSCs before transplantation.
This is a 2020 ARVO Annual Meeting abstract.