Comparative analysis of different feeder layers with 3T3 fibroblasts for culturing rabbits limbal stem cells

Abstract

Aim: To explore the possibility of human umbilical cord mesenchymal stem cells (hUCMSCs), human umbilical vein endothelial cells (hUVECs), human dental pulp stem cells (hDPSCs) and human periodontal ligament stem cells (hPDLSCs) serving as feeder cells in co-culture systems for the cultivation of limbal stem cells.

Methods: Different feeder layers were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 and were treated with mitomycin C. Rabbits limbal stem cells (LSCs) were co-cultured on hUCMSCs, hUVECs, hDPSCs, hPDLSCs and NIH-3T3, and then comparative analysis were made between each group to see their respective colony-forming efficiency (CFE) assay and immunofluorescence (IPO13,CK3/12).

Results: The efficiency of the four type cells in supporting the LSCs morphology and its cellular differentiation was similar to that of NIH-3T3 fibroblasts as demonstrated by the immunostaining properties analysis, with each group exhibiting a similar strong expression pattern of IPO13, but lacking CK3 and CK12 expression in terms of immunostaining. But hUCMSCs, hDPSCs and hPDLSCs feeder layers were superior in promoting colony formation potential of cells when compared to hUVECs and feeder-cell-free culture.

Conclusion: hUCMSCs, hDPSCs and hPDLSCs can be a suitable alternative to conventional mouse NIH-3T3 feeder cells, so that risk of zoonotic infection can be diminished.

Keywords: dental pulp stem cells; feeder layers; limbal stem cells; periodontal ligament stem cells; umbilical cord mesenchymal stem cells; umbilical vein endothelial cells.

Figure 1. Morphology of hUCMSCs, hUVECs, hDPSCs and hPDLSCs

A: P4 of hUCMSCs (×50); B: P2 of hUVECs (×100); C: P3 of hDPSCs (×100); D: P3 of hPDLSCs (×100).

Figure 2. MTT assay test the influence of different concentration of mitomycin C for the cell proliferation rates

A: Influence on the hUCMSCs; B: Influence on the hPDLSCs.

Figure 3. Morphology and growth profile of freshly collected LSCs (P0)

The LSCs cultivated with hUCMSCs (A), hUVECs (B), hDPSCs (C), hPDLSCs (D) and NIH-3T3 (E) on day 8 (×100).

Figure 4. Cellular localization of LSC markers by fluorescent immunostaining

LSCs on three types of feeder layer were subjected to immunostaining against IPO13 (A-E). DAPI depicts cell nuclei (F-J). Merged images were shown in (K-O). A, F, K: LSCs on hUCMSCs; B, G, L: LSCs on hUVECs; C, H, M: LSCs on hDPSCs; D, I, N: LSCs on hPDLSCs; E, J, O: LSCs on 3T3 fibroblasts.

Figure 5. Cellular localization of LSC markers by fluorescent immunostaining

LSCs on three types of feeder layer were subjected to immunostaining against CK3/12 (A-E). DAPI depicts cell nuclei (F-J). Merged images are shown in (K-O). A, F, K: LSCs on hUCMSCs; B, G, L: LSCs on hUVECs; C, H, M: LSCs on hDPSCs; D, I, N: LSCs on hPDLSCs; E, J, O: LSCs on 3T3 fibroblasts.