Fig 1.
Generation of Neural Crest Cells (NCCs) from human induced Pluripotent Stem Cells (hiPSCs).
(A) hiPSC colonies were observable at day 0 of a 12-day protocol for Neural Crest Cells (NCCs) derivation. On day 9, cells were more mesenchymal in appearance and possessed a typical NCC-like dendritic morphology. (B)(C) We observed hiPSCs were SOX2+, but hiPSC derived NCCs were SOX2-. >80% of derived NCCs were positive for neural crest markers AP2a and p27NTR. Brightfield scale bar represents 200 μm, low magnification (10X) confocal images scale bar represents 100 μm, high magnification (63X) confocal images scale bar represents 10 μm.
Fig 2.
Expression analysis of derived NCCs compared to hiPSCs.
Quantitative polymerase chain reaction (qPCR) analysis showed derived NCCs had higher expression of many neural crest markers relative to hiPSCs. The pluripotency markers SOX2, OCT3/4 and NANOG were significantly reduced in NCCs, but KLF4 expression was maintained at a similar level.
Fig 3.
3D culturing promotes a corneal keratocyte-like cell fate.
(A) Panels show hiPSC-derived NCCs stained for HNK1, Vimentin, AP2a, Keratocan (shown in red as it was co-stained with the AP2a panel) and ABCB5. (B) 3D cultures were generated as shown in the schematic at the top of the Figure. Panels show transverse cryosections of the 3D cultured NCCs stained for HNK1, Vimentin, AP2a, Keratocan (red) and ABCB5. Arrows in the Keratocan+ panel indicates extracellular deposits of Keratocan. Scale bar represents 100 μm.
Fig 4.
Expression analysis of 3D-cultured NCCs compared to non 3D-cultured NCCs and cadaveric human corneal keratocytes.
(A) qPCR analysis showed 3D cultured derived NCCs had lower levels of expression of many neural crest markers, such as PAX3, SOX10, CX32, FOXD3 and MSX1, compared to non-3D cultured NCCs. Genes that are highly expressed in corneal keratocytes were up-regulated by 3D culture. (B) The gene expression profile of 3D-cultured NCCs was largely incompatible with cadaveric corneal keratocytes. Most genes assayed had significantly higher or lower levels of gene expression when the two populations were compared.
Fig 5.
hiPSC derived NCCs seeded onto limbal rims migrate to the cornea and then migrate into the stroma.
(A) hiPSC-derived NCCs were seeded onto the scleral region of limbal rim slices as shown in the schematic. (B) After 4 days, live cell staining with Calcein AM indicated all derived NCCs had migrated to the corneal surface region of the limbal rim slice. By day 7 derived NCCs had migrated into the stroma of the cornea. Scale bars for low magnification images (let hand side) represent 200 μm and for higher magnification images (right hand side) represent 100 μm.
Fig 6.
Limbal rim seeded NCCs adopt a corneal keratocyte-like fate and migrate between the collagen lamellae.
(A) Control non-seeded rim cryosections showed no DAPI+ nuclei or AP2a+/ Keratocan+ cells. Limbal rims seeded with derived NCCs did contain AP2a+/ Keratocan+ cells after 21 days of culture (arrows). (B) High magnification images show Keratocan+ derived NCCs migrated within the collagen lamellae of the stromal layer of the cornea (this panel is a higher magnification of the Keratocan staining shown in (A). ABCB5+, Vimentin+ and HNK1+ staining was also detected in these cells. Scale bars in (A) represent 100 μm, scale bars in (B) represent 50 μm.
Fig 7.
Expression analysis shows limbal rim seeded NCCs adopt an expression profile that is similar to human corneal keratocytes.
qPCR analysis indicated hiPSC-derived NCCs cultured on limbal rim slices (blue bars) adopted an expression profile that was more analogous to human corneal keratocytes than hiPSC-derived NCCs grown in 3D cultures (red bars). Importantly, the expression of a number of keratocyte markers was much higher when derived NCCs were cultured on limbal rim slices, such as ALDH3A1, KERATOCAN, PtGDS and AQP1.