Discovering the Potential of Dental Pulp Stem Cells for Corneal Endothelial Cell Production: A Proof of Concept

Abstract

Failure of corneal endothelium cell monolayer is the main cause leading to corneal transplantation. Autologous cell-based therapies are required to reconstruct in vitro the cell monolayer. Several strategies have been proposed using embryonic stem cells and induced pluripotent stem cells, although their use has ethical issues as well as limited clinical applications. For this purpose, we propose the use of dental pulp stem cells isolated from the third molars to form the corneal endothelium cell monolayer. We hypothesize that using dental pulp stem cells that share an embryological origin with corneal endothelial cells, as they both arise from the neural crest, may allow a direct differentiation process avoiding the use of reprogramming techniques, such as induced pluripotent stem cells. In this work, we report a two-step differentiation protocol, where dental pulp stem cells are derived into neural crest stem-like cells and, then, into corneal endothelial-like cells. Initially, for the first-step we used an adhesion culture and compared two initial cell sources: a direct formation from dental pulp stem cells with the differentiation from induced pluripotent stem cells. Results showed significantly higher levels of early stage marker AP2 for the dental pulp stem cells compared to induced pluripotent stem cells. In order to provide a better environment for neural crest stem cells generation, we performed a suspension method, which induced the formation of neurospheres. Results showed that neurosphere formation obtained the peak of neural crest stem cell markers expression after 4 days, showing overexpression of AP2, Nestin, and p75 markers, confirming the formation of neural crest stem-like cells. Furthermore, pluripotent markers Oct4, Nanog, and Sox2 were as well-upregulated in suspension culture. Neurospheres were then directly cultured in corneal endothelial conditioned medium for the second differentiation into corneal endothelial-like cells. Results showed the conversion of dental pulp stem cells into polygonal-like cells expressing higher levels of ZO-1, ATP1A1, COL4A2, and COL8A2 markers, providing a proof of the conversion into corneal endothelial-like cells. Therefore, our findings demonstrate that patient-derived dental pulp stem cells may represent an autologous cell source for corneal endothelial therapies that avoids actual transplantation limitations as well as reprogramming techniques.

Keywords: cell reprogramming; cornea; corneal endothelium; dental pulp stem cells; differentiation; neural crest.

Figure 1

Schematic representation of corneal endothelial differentiation based on the pluripotency levels. On the one hand, somatic cells as fibroblasts and DPSC can be reprogrammed into iPSC, which can be differentiated into NCSC and then into CEC. On the other hand, DPSC could be dedifferentiated into NCSC, and then differentiated into CEC.

Figure 2

Cell reprogramming from DPSC and fibroblasts. Relative mRNA expression in DPSC and fibroblasts (positive control), before and after cell reprogramming (A). Pluripotent expression of Oct 4 and Nanog by quantitative RT-PCR. Optical microscope images of iPSC colonies cultured in VTN-coated dishes from fibroblasts (positive control) and from DPSC (B). *Scale bars: 200 μm (lower magnification) and 100 μm (higher magnification). **Data presented are means ± SD, n = 3 technical replicates per donor and experimental group. Different letter denotes significant differences (p < 0.05). Same letter denotes non-significant differences.

Figure 3

Formation of NCSC using an adherent method. Microscope images of NCSC-derived from iPSC and from DPSC at day 21 of differentiation (A). Relative mRNA expression in NCSC-derived from DPSC and iPSC, at days 0 and 21, analyzed by quantitative RT-PCR (B). *Scale bar: 100 μm. **Data presented are means ± SD, n = 3 technical replicates per experimental group. Different letter denotes significant differences (p < 0.05). Same letter denotes non-significant differences.

Figure 4

Formation of NCSC from DPSC using a suspension method. Microscope images of cell morphology at days 1, 2, 3, 4, and 5 of NCSC formation in suspension culture (A). Neurosphere diameter at days 1–5 of suspension culture (B). *Scale bars: 500 μm (lower magnification) and 250 μm (higher magnification). **Data presented are means ± SD. Different letter denotes significant differences (p < 0.05). Same letter denotes non-significant differences.

Figure 5

Gene expression of NCSC formation from DPSC in suspension culture. Relative mRNA expression of NCSC markers AP2, Nestin, and p75 (A) and pluripotent markers Oct4, Nanog, and Sox2 (B) analyzed by quantitative RT-PCR. *Data presented are means ± SD, n = 3 technical replicates. Different letter denotes significant differences (p < 0.05). Same letter denotes non-significant differences.

Figure 6

Expansion of NCSC from DPSC. Microscope images of NCSC-derived from DPSC at days 4, 12, and 19 (A). Relative mRNA expression in NCSC-derived from DPSC, at days 0, 4, and 19 of NCSC markers AP2, Nestin, and p75 (B) and of pluripotent markers Oct4, Nanog, and Sox2 (C), by quantitative RT-PCR. *Scale bars: 500 μm (lower magnification) and 200 μm (higher magnification). **Data presented are means ± SD, n = 3 technical replicates. Significant differences compared to day 0 (p < 0.05).

Figure 7

CEC differentiation from DPSC. Microscope images of CEC-derived from DPSC at days 12 and 19 (A). Relative mRNA expression in CEC-derived from DPSC, at days 0, 4, and 19 of CEC markers ZO-1, ATP1A1, COL4A2, and COL8A2, by quantitative RT-PCR (B). *Scale bars: 250 μm (lower magnification) and 100 μm (higher magnification). **Data presented are means ± SD, n = 3 technical replicates per experimental group. Different letter denotes significant differences (p < 0.05). Same letter denotes non-significant differences.