Induction of perioculer neural crest markers in developing corneal endothelium form iPS cells

Abstract

Purpose: We are now developing regenerative medicine of corneal endothelium (CE) cells form hum induced pluripotent stem (iPS) cells. CE is developed from pericular neural crest (PNC) cells delivered from neural crest (NC) cells. We have reported retinoic acid (RA) is a key factor which induce a PNC marker, PITX2. In this research, we investigated the effect of RA to other perioculer NC markers.

Methods: iPS cells (253G1) were cultured for weeks by sphere culture without serum, and NC cells were isolated by FACS using p75NTR as a marker. Then isolated NC cells were seeded on dishes with retinoic acid in 0.1uM and 1uM concentrations. The cells were harvested and the expression of PNC markers, PITX2, FOXC1 and FOXC2 was quantified by RT-PCR method. To increase the level of the markers, additional wnt3A application was tested.

Results: PITX2 expression level showed a peak 5 to 10 days after application of RA. (<0.01% of GAPDH expression level before application, 1.92% of that in 10 days after application, respectively). FOXC1 expression level was also increased at 10 days after application (16.41 % of GAPDH expression level, which is same level in cultured CE cells). However, another marker FOXC2 was not induced. Additional wnt3A application increase the FOXC2 expression level at 10 days.

Conclusions: RA and wnt signals are important in the cell fate determination from NC cells to perioculer NC cells

Keywords: 481 cornea: endothelium • 480 cornea: basic science