Modulation of Rabbit Corneal Epithelial Cells Fate Using Embryonic Stem Cell Extract

Weijiao Zhan, Zhiping Liu, Ying Liu, Qicheng Ke, Yuanyuan Ding, Xiaoyan Lu, Zhichong Wang

Introduction

Conjunctivalization of the cornea and subsequent vision loss is an effect of corneal damage combined with limbal stem cell deficiency. Limbal stem cells provide an option for cell therapy; however, they are difficult to isolate and expand in an effective manner (can take a long time). The dedifferentiation or reprogramming of adult somatic cells provides another means to attain patient-specific stem cells for tissue regeneration. Previous studies have shown that embyronic stem cell (ESC)-derived cell-free factors and proteins are capable of reprogramming somatic cells into pluripotent cells without the use of nuclear transfer or transfection of reprogramming genes. The goal of this study was to develop a culture system to culture and dedifferentiate autologous somatic cells (rabbit corneal epithelial cells) into pluripotent cells for use in cell therapy and tissue engineering.

Methods

Cells
For this study, mouse ESC cell line ES-E14 was cultured and used for the extraction of embryonic stem cell extract. The cells were plated on 1% gelatin on tissue-treated culture plates with ESC culture media, where 50% of the media was changed every day. The ES-E14 cells were stained with anti-mouse Oct-4 antibody to confirm the undifferentiated state of the ESCs. Primary corneal epithelial cells were cultured from rabbit peripheral corneal tissue explants on tissue-treated culture plates with corneal epithelial medium.

Cell Extract Preparation
Cells were washed using PBS and cell lysis buffer, followed by centrifugation at 400x g, and resuspended with cell lysis buffer with a 40 minute incubation on ice. The cells were sonicated on ice until all cells and nuclei had been lysed. The lysate was centrifuged at 15000x g for 15 min at 4 degrees C. The supernatant was transferred to a 15 mL tube and snap-frozen and stored at -80 degrees C to remove any remaining living cells.

Cell Extract Treatment
The primary rabbit corneal epithelial cells were permeabilized using streptolysin-O (SLO) for 15 min at 37 degrees C with constant agitation. The cells were then resuspended in mES-cell extract, along with ATP, creatine phosphate, creatine kinase, and NTP, and incubated at 37 degrees C in a water bath with constant agitation. The cell membranes were resealed by transfer to epithelial cell culture media with CaCl2 and seeded onto a tissue culture plate for 2 hours at 37 degrees with 5% CO2. The media was then replaced with complete epithelial cell culture media and changed every other day. Colonies were isolated and transferred to ESC medium for culture.

Histology and Immunohistochemistry
This was performed after 24-48 hours of culture for Oct-4, SSEA1, K3/K12, p63, ABCG2, and Vimentin. They were counterstained with Hoechst 33342, followed by imaging using a confocal laser scanning microscope. The cells were also tested for gene expression using RT-PCR, stained with BCIP/NBT Phosphate Substrate System to test for AKP activity, and tested for tumorigenicity (teratoma formation after subcutaneous injection).

Results
The results indicated mES-colony like structures in passage 5 or week 3 of culture. The colonies were maintained in ESC medium for at least 14 weeks in culture, but began to flatten out. Oct-4 expression was detected in P2, and peaked at P9 (week 4), decreasing in subsequent weeks. SSEA1 was detected in P9, but not in P18, and neither of these markers were detected in the control samples. K3 and p63, which are specific for corneal tissue and progenitor cells, were also detected in the cells, indicating that the cells were not completely reprogrammed to and ES state, but were still capable of returning to the start of their lineage. Vimentin was not detected, indicating a lack of fibroblast contamination. ESC extract induced cells formed teratomas and were capable of differentiating into all three germ layers. AKP staining revealed positive staining at P9, but weaker staining at P18

Figure 1

Representative phase-contrast micrographs of cells. A-C: SLO+ corneal epithelial cell extract-induced cells (p-Pc) P1 (day 8), P3 (day 12), P5 (day 14); D-F: cells without any treatment (b-Pc) P1 (day 3), P3 (day 9), P5 (day 14); G-I: SLO+ES cell extract-induced cells (e-Pc) P1 (day 8), P3 (day 12), P5 (day 14); J-L: e-Pc P9 (wk 4), P12(wk 6), P18 (wk 8). The magnification of panel E is 50×, the other panels are at 100×. The three groups maintained similar morphology at P1 (day 8; A, D, and G). Some fibroblast-like cells took place in p-Pc group (B and C). The P3 cells in b-Pc control exhibited aging appearance with vacuoles and larger size, and could not survive through P5 (E and F). At P3 (day 14) of e-Pc, among the aging epithelial cells, colonies with small cells and vague boundaries showed up (H). At P5 (week 3), colonies that closely resembled to mES colonies could be readily identified by their morphology (I). This phenomenon was maintained for at least 14 weeks in culture, corresponding to 26 passages, but the colonies gradually became flat (J-L).

Figure 2

mRNA expression of Oct-4, K3, and p63 with GAPDH as an internal control for P2 in all groups, E₁₄ and P6, P9, P18 of e-Pc. After the mES cell extract treatment, Oct-4 mRNA was detected in P2 (day 12), reached its peak at P9 (week 4), and decreased in later passages. It remained undetectable in the two control groups. Expression of corneal tissue-specific marker K3 mRNA increased as passage in experiment group, and progenitor cell marker p63 was also found in these cells.

Figure 3

Expression of pluripotency-associated proteins Oct-4 and SSEA1 in e-Pc with immunofluorescent staining. The scale bar represents 50 μm. Oct-4 and SSEA1 proteins were found in P9 (week 4), not in P18 (week 8) cells.

Figure 4

Expression of corneal-epithelium-related proteins in different conditions and passages with immunofluorescent staining. A-C: p63, ABCG2, and K3 (green, positive cells; blue, nuclei) for P2 in all groups and P9, P18 of e-Pc. D: Vimentin for P6 in p-Pc group. E: Vimentin for P9 in e-Pc group. The scale bar in B represents 50 μm. Corneal tissue-specific marker K3 and progenitor cell markers, p63 or/and ABCG2 were still found in different passages of e-Pc. Vimentin, an intermediate filament protein and a characteristic of keratocytes and fibroblasts was not detected in P9 cells of e-Pc. But it was positive in P6 of p-Pc.

Figure 5

Teratoma formation examination and alkaline phosphatase (AKP) staining. A: HE staining shows teratoma from ES cells and P9 (wk 4) e-Pc containing multiple tissues, including epithelium (E), neural (N), muscle (M), and glandular structures (G). The scale bar represents 50 μm. B: AKP staining in P9 (wk 4) and P18 (wk 8) e-Pc (magnification, 100×). AKP staining was positive at P9 (week 4), and positive but weaker at P18 (week 8). Small and cohesive colonies were mostly observed in P9 cells. Flatter, larger and more migratory colonies were noted in P18 cells.


Critique
The paper shows that embyronic stem cell extract is capable of dedifferentiating rabbit corneal epithelial cells; however, the cells are incapable of maintaining this state and can only be partially reprogrammed to multipotency. It was also demonstrated they can be quickly dedifferentiated, expanded, and maintained for many weeks. Although this study has demonstrated that embryonic cell extract, the study did not demonstrate that there is global expression of precursor and embryonic markers. Also, there was no study done to demonstrate that these partially dedifferentiated cells were capable of achieving similar functionality as rabbit embryonic stem cells and corneal epithelial cells. The paper was pretty thorough in testing both qualitatively through immunostaining as well as quantitatively through RT-PCR, and is a good first step; however, these cells need to be compared to other cells (ES cells, progenitor cells, corneal epithelial) to determine their functionality. After this functionality has been determined, in vivo studies regarding recovery of vision loss and prevention of conjunctivalization of the cornea need to be performed.