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Pair-cell analysis was performed as described (Shen et al., 2002 (link)). Following electroporation at E14.5 as described above, electroporated brains at E16.5 and E17.5 were sectioned using a vibratome (Leica, VT1000S). Cortical tissues that were enriched for transferred cells (marked by GFP) were dissected with tungsten needles from cortical sections. For the cortex transfected with Mek1^DD, the upper and lower half of the cortex were further separated. To dissociate cells, dissected tissues were incubated in a protease solution containing 10 unit/ml papain (Fluka, Japan), 1000 units/ml DNAse I (Roche, Switzerland) and 5 mM L-cysteine in DMEM (Invitrogen), and triturated using a fire-polished Pasteur pipette to create a single-cell suspension. Cells were resuspended in culture medium containing DMEM, glutamine, penicillin/streptomycin, sodium pyruvate (Invitrogen), 1 mM N-acetyl-L-cysteine (Sigma), B27, N2 and 10 ng/ml bFGF2 (Invitrogen) and plated onto coverslips coated with poly-L-lysine (Sigma) at clonal density. The cultures were maintained in a humidified incubator at 37°C with constant 5% CO₂ supply. In 24 or 48 hr later, the cultures were fixed and immunostained for GFP, Fabp7, and Tubb3 and counterstained with the DNA dye Hoechst 33342 solution (Invitrogen).

Keratocyte differentiation of PDLSCs was induced with keratocyte differentiation medium (KDM) as described by Syed-Picard and collaborators [11 (link)]. KDM was prepared in advanced DMEM (Life technologies, 12491) supplemented with 1 mM A2-P, 10 ng/mL of basic fibroblast growth factor-2 (bFGF-2; Invitrogen, PHG6015) and 0.1 ng/mL of transforming growth factor-beta3 (TGF-β3; Sigma-Aldrich, SRP3171). After seeding, PDLSCs were cultured in growth medium for 2 days before switching to KDM. The differentiation medium was changed every 2 days. To evaluate the differentiation efficacy of KDM on PDLSCs, cultured primary human limbal keratocytes (at passage 2) were used as controls in the qPCR assay. Primary human limbal keratocytes were isolated and cultured, and have been characterized by our group [25 (link)]. Briefly, corneal epithelial and endothelial cells were removed by scraping. The remaining limbal corneal stroma was cut into pieces and digested with collagenase overnight. After centrifugation, the cells were cultured in DMEM/F-12 (Gibco, #21331-046) supplemented with 2% FBS and 1% penicillin-streptomycin. Cells at passage 2 were collected for RNA extraction and followed by qPCR assay.
To evaluate the effect of substance P on keratocyte differentiation, PDLSCs were cultured in KDM with or without 1 μM SP (Sigma, S6883). The medium was changed every second day.

Culture cells were harvested in trypsin-EDTA and carefully resuspended in DMEM/F-12 medium (Lonza, Walkersville, MD) supplemented with 1% N2 supplement, 2% B27 supplement, 20ng/ml b-FGF-2 and 20ng/ml EGF (GIBCO Life Technologies, NY, USA). Around 5000 cells were counted and plated in each well of a six-well ultra-low-attachment plate (Corning, Lowell, MA). Orospheres were assayed 7 to 14 days after cell plating. Orospheres were counted manually under a light microscope, and experiments were done in triplicates.