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To evaluate the effect of both direct celltocell contact with feeder cells and soluble factors secreted by feeder cells, 3T3 fibroblasts feeder cells were seeded on a cell culture inserts at a density of 2.5×104/cm2. On the following day, 3×105 epithelial cells were seeded on the insert. After epithelial cells reached confluence, 2.5×104/cm2 3T3 feeder cells were seeded again in the bottom of the well. On the following day, epithelial cells were airlift cultured for an additional week to allow stratification (Compound feeder culture, Figure 1C).
As a control, 3×105 epithelial cells were seeded on the insert. After epithelial cells reached confluence, cells were airlift cultured for an additional week to allow stratification (Culture without feeder cells, Figure 1D).
Histological Observation and Immunostaining Fresh frozen cryosections were fixed with Formalin and stained with hematoxylin and eosin (HE) for histological observation.
For immunohistochemistry, fresh frozen cryosections were fixed with 40g/L paraformaldehyde (PFA, Wako Ltd. Osaka, Japan). PFAfixed cells were permeabilized with 3g/L Triton X100 (SigmaAldrich, St. Louis, MO). After background staining was blocked with 100mL/L normal donkey serum, the cells were treated with the following monoclonal primary antibodies: antip63 (1∶100, 4A4; Santa Cruz biotechlogy, inc, Santa Cruz, CA), antiK19 (NeoMarkers For Lab Vision Corporation, Fremont, CA), and antiinvolucrin (1∶100, Covance, Emeryville, CA). The cells were then treated with Cy3conjugated secondary antibodies (Chemicon International, Inc., Temecula, CA). The nuclei were counterstained with 4, 6diamino2phenylindole (1mg/mL, DAPI; Dojindo Laboratories, Tokyo, Japan).
RESULTS
Stratification was limited to three to four layers in the contact feeder group (Figure 2A), whereas separate feeder sheets were slightly more stratified (Figure 2B). However, the compound feeder group revealed marked stratification and produced a stratified epithelium with five to seven layers of cells (Figure 2C). As a control, the group without 3T3 feeder cells was the least stratified and formed only two to three layers of cells (Figure 2D).
Immunostaining images in the compound feeder group showed expression of progenitor markers p63 (Figure 3A) and K19 (Figure 3B) in the basal and suprabasal layer, as well as differentiation marker involucrin (Figure 3C) in the whole layer.
DISCUSSION
Epithelialmesenchymal interactions control epidermal growth and differentiation and regulate tissue homeostasis in the epidermis[4,5]. Feeder cells support the serial subculture of epithelial cells though several passages[6], allow colony formation by epithelial cells[4,7], inhibit the growth of contaminating fibroblasts[6], and maintain human telomerase reverse transcriptase expression[8] and Sp1/Sp3 activity[9]. This interplay is based on two basic processes: production of Figure 3Immunostaining images of mouse corneal epithelial sheets Expression of progenitor markers p63 (A) and K19 (B) was located in the basal and suprabasal layer, as well as differentiation marker involucrin (C) in the whole layer.
Scale bars, 50μmsoluble factors displaying autocrine and paracrine activities[4], and direct cellcell/matrix contact[1012]. However, their functional significance in the dermalepidermal interplay to regulate epithelial stratification and homeostasis is only poorly understood.
In our study, we compared four culture methods to investigate the roles of direct contact and soluble factors in stratification of murine corneal epithelial cells. The results showed that the compound feeder culture produced the most robust epithelial sheets with five to seven layers of cells (Figure 2C), both contact and separate feeder culture formed three to four layers(Figure 2A and 2B), however, the group without 3T3 feeder cells was the least stratified and limited to only two to three layers of cells (Figure 2D). The results suggested that direct contact as well as production of soluble factors play important roles in epithelial stratification and homeostasis. The compound feeder culture took the advantages of both of them and revealed marked stratification.
The expression of p63[13,14] and K19[15,16] was localized to progenitor cells with high proliferative capacity, including both limbal stem cells (LSCs) and transient amplifying cells (TACs) at present. Immunostaining showed in the compound feeder group the expression of progenitor markers p63 (Figure 3A) and K19 (Figure 3B) in the basal and suprabasal layer, which indicates that the cells in the basal and suprabasal layer of the sheet still maintained the phenotype of corneal epithelial progenitor cells and had high proliferative capacity. Involucrin[17] have been regarded as markers of differentiation of the corneal epithelial cells. The cells in the epithelial sheets expressed involucrine, which means that the cells retained the potential of differentiation.
In conclusion, the remarkable stratification as well as the limbal phenotype makes the compound feeder system a candidate tool for cultivating transplantable epithelial sheets.
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