Experimental Study of the Biological Properties of Human Embryonic Stem Cell-Derived Retinal Progenitor Cells

Abstract

Retinal degenerative diseases are among the leading causes of blindness worldwide, and cell replacement is considered as a promising therapeutic. However, the resources of seed cells are scarce. To further explore this type of therapy, we adopted a culture system that could harvest a substantial quantity of retinal progenitor cells (RPCs) from human embryonic stem cells (hESCs) within a relatively short period of time. Furthermore, we transplanted these RPCs into the subretinal spaces of Royal College of Surgeons (RCS) rats. We quantified the thickness of the treated rats' outer nuclear layers (ONLs) and explored the visual function via electroretinography (ERG). It was found that the differentiated cells expressed RPC markers and photoreceptor progenitor markers. The transplanted RPCs survived for at least 12 weeks, resulting in beneficial effects on the morphology of the host retina, and led to a significant improvement in the visual function of the treated animals. These therapeutic effects suggest that the hESCs-derived RPCs could delay degeneration of the retina and partially restore visual function.

Figure 1. Cell cycle analysis of differentiated cells at 0, 10, 20, 30, and 40 days.

(A to E) Cell cycle showed that the mitotic ratio was highest at day 0 of differentiation and then decreased. (F) Data of cell cycle at different times were shown as mean ± standard deviation (SD) in the form of table. (G to H) Statistical analysis showed the mitotic ratio of cells at different times. Data from at least three independent experiments are represented as the mean ± SD. All data expressed as mean ± SD. ***P < 0.001 versus with data of day 0.

Figure 2. hESCs differentiated toward a RPC phenotype.

(A) H1 differentiated into a RPC and expressed cell-specific markers—Pax6, Sox2, Rax, and Nestin, evaluated by Immunofluorescence. (B to E) Staining of Pax6, Sox2, Rax, and Nestin was evaluated by using FACS. Blue line, isotype. (F) Statistical analysis of expression of Pax6, Sox2, Rax, and Nestin. Cell nuclei are shown in 6-diamidino-2-phenylindole (DAPI), blue. Data from at least three independent experiments are represented as the mean ± SD. Scale bars = 25 μm. *P < 0.05, **P < 0.01, ***P < 0.001, versus with data of previous time point.

Figure 3. hESCs differentiated toward a PPC phenotype.

(A to C) H1 differentiated into a PPC and expressed cell-specific markers—Otx2, Crx, and Recoverin, examined by FACS. (D) Statistical analysis of expression of Otx2, Crx, and Recoverin. Data from at least three independent experiments are represented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, versus with data of previous time point.

Figure 4. The expression level of proteins and genes of retinal progenitor cells during the differentiated development of hESCs analyzed by Western blot and real-time PCR.

(A) Western blot analysis of retinal progenitor cell markers (Pax6, Sox2, Rax, Nestin, Otx2, Crx, and Recoverin) during their differentiated development. The gels in this experiment were run under the same experimental conditions. (B) Statistical analysis of the expression levels of proteins in retinal progenitor cell markers (Pax6, Sox2, Rax, Nestin, Otx2, Crx, and Recoverin). Data expressed as mean ± SD. (C) Statistical analysis of the expression of mRNA in retinal progenitor cell markers (Pax6, Sox2, Rax, Nestin, Otx2, Crx, and Recoverin). Data from at least three independent experiments are represented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, versus with data of previous time point.

Figure 5. CM-Dil–labeled transplanted cells (red) survived in the host retina and migrated into the ONL.

Micrographs represented the superior region of the retina. (A to C) showed the surviving cells in the images at 4 weeks (A), 8 weeks (B), and 12 weeks (C) after transplantation. (A’ to C’) magnified images of the small white rectangles in the centers showing cells integrated into the ONL. (D) The number of surviving cells in a section decreased as the time since transplantation grew longer. Data from at least three independent experiments are represented as the mean ± SD. *P < 0.05, versus with data of previous time point. Cell nuclei are shown in blue with DAPI; transplanted cells are shown in red; white arrowheads indicate surviving cells in the ONL and subretinal space. GCL, ganglion cells layer; INL, inner nuclear layer; ONL, outer nuclear layer; SRS, subretinal space. A to C scale bars = 100 μm; A’ to C’ scale bars = 25 μm.

Figure 6. The micrographs showing the thicknesses of the ONL across the vertical meridian.

(A1 to A3) Retinas of Long-Even rats at 4 weeks, 8 weeks, and 12 weeks. The average ONL thicknesses were 44.74 ± 1.31 μm, 44.84 ± 1.92 μm, and 45.82 ± 3.29 μm respectively. (B1 to B3) Retinas of sham-treated rats at 4 weeks, 8 weeks, and 12 weeks. The average ONL thicknesses were 7.72 ± 1.01 μm, 6.71 ± 0.52 μm, 4.22 ± 0.73 μm respectively. (C1 to C3) Retinas of transplanted rats at 4 weeks, 8 weeks, and 12 weeks. The average ONL thicknesses were 28.60 ± 1.84 μm, 23.32 ± 0.84 μm, 19.82 ± 1.18 μm respectively. (D) Statistical analysis of ONL thicknesses in three groups at corresponding time. Data from at least three independent experiments are represented as the mean ± SD. GCL, ganglion cells layer; INL, inner nuclear layer; ONL, outer nuclear layer; SRS, subretinal space. ***P < 0.001 compared with the wild-type group; ^oooP < 0.001 compared with the sham-treated group. Cell nuclei are shown in blue; transplanted cells are shown in red. Scale bars = 100 μm.

Figure 7

Waveforms of ERG at 4 (A), 8 (B), and 12 (C) weeks after transplantation. (A to C) The transplanted group did better on oscillography than the sham-treated group at 4 weeks, 8 weeks, and 12 weeks post-transplantation. (D) Statistical analysis of average b-wave amplitude of eyes in the transplanted group as compared with those in the sham-treated group 4, 8, and 12-weeks after transplantation. Data from at least three independent experiments are represented as the mean ± SD. *P < 0.05 compared with the sham-treated group.

Figure 8. Teratoma assay of hESC-derived RPCs in SCID mice.

(A) Teratomas were not observed in the hESC-derived RPC group. (B) Teratomas formation were detected in 2 of 6 SCID mice in the hESC group. (C) The proportion of hESC-derived RPCs and hESCs in the teratoma assay. (D) A teratoma derived from hESCs group. Scale bar = 0.5 cm.