Generation, purification and transplantation of photoreceptors derived from human induced pluripotent stem cells

Abstract

Background: Inherited and acquired retinal degenerations are frequent causes of visual impairment and photoreceptor cell replacement therapy may restore visual function to these individuals. To provide a source of new retinal neurons for cell based therapies, we developed methods to derive retinal progenitors from human ES cells.

Methodology/physical findings: In this report we have used a similar method to direct induced pluripotent stem cells (iPS) from human fibroblasts to a retinal progenitor fate, competent to generate photoreceptors. We also found we could purify the photoreceptors derived from the iPS cells using fluorescence activated cell sorting (FACS) after labeling photoreceptors with a lentivirus driving GFP from the IRBP cis-regulatory sequences. Moreover, we found that when we transplanted the FACS purified iPSC derived photoreceptors, they were able to integrate into a normal mouse retina and express photoreceptor markers.

Conclusions: This report provides evidence that enriched populations of human photoreceptors can be derived from iPS cells.

Figure 1. Co-expression of undifferentiated cell markers Sox2 (green, A′), SSEA-4 (blue, A″) and Oct4 (red, A″′) by iPS cells with merged view in (A).

(B-D) Tri-lineage potential of teratomas formed by iPSC-MHF2 c1 in immunodeficient mice. Histological sections from a teratoma stained with antibodies against human MAP-2 (microtubule associated protein-2, B), human SMA (alpha smooth muscle actin, C), or human AFP (alpha-fetoprotein) and co-stained with DAPI (blue).

Figure 2. (A) RT-PCR comparison of expression of EFTFs and CRX in the various human ES and iPS cell lines expressed as a cycle change in PCR expression level compared to undifferentiated cells (n = 3–6) for the various lines, error bars represent SEM.

(B) Gel showing expression of retinal markers by RT-PCR following retinal induction of iPS cells. (C) Co-expression of Pax6 (white, C, C′), Sox2 (green, C, C″), and Sox9 (red, C, C″′) by iPS cell derived retinal progenitors (merged in (C)) at the end of three weeks. The progenitors also expressed nestin (white, D,D') in the typical rosette pattern with ZO-1(green, D, D″) in the center and Brn3 expressing ganglion cells (red, D, D″′) at the periphery. (E, F) RT-PCR graph showing time course of expression of various retinal progenitor cell (E) and photoreceptor cell (F) genes over the course of 5 weeks. Error bars represent SEM.

Figure 3. Cells in culture at two months after initiation of the protocol expressed HuC/D (green, A) marking ganglion and amacrine cells and Nrl (red, A) which labels rod photoreceptors.

Photoreceptors were identified by the expression of Otx2 (green, B), Crx (E), recoverin (red, C) and AIPL1 (red, D). Cells also expressed differentiated markers like rhodopsin (red, G) and S-opsin (green, F). (H) Retinal pigmented epithelium was also generated from the iPS cells and these cells expressed ZO-1 (inset). Nuclei stained with DAPI in blue.

Figure 4. IRBP-GFP infection of human retinal explant resulted in GFP expression in photoreceptors as evidenced by co-expression of Otx2 (red, A, A″), AIPL1 (white, A, A″′), recoverin (red, B, B″) and Blimp1 (white, B, B″′).

IRBP-GFP expression in iPS cell-derived retinal cells. GFP cells co-expressed AIPL1 (red, C, C'), Nrl (red, D, D'). Nuclei stained with DAPI in blue.

Figure 5. Microarray analysis of FACS sorted human fetal retinal IRBP-GFP cells and 96 day unsorted fetal retinal cells.

The figure shows the heat map of the spot intensity comparison on the microarray of the various photoreceptor and retinal progenitor genes.

Figure 6. Fluorescent activated Cells sorting of human cells.

Following FACS and fixation, upon immunostaining, GFP cells (A) expressed Crx (red, B). (C) Representative FACS plots for GFP cells in BD Aria II. (D) Plot showing percentage of sorted cells expressing Crx and recoverin.

Figure 7. Transplantation of FACS IRBP-GFP human iPS cells inot wild-type mouse retina.

(A) shows the photoreceptor layer stained for recoverin (red), Otx2 (white) and DAPI (blue). GFP expressing transplanted cells have migrated in and also express these markers. Also shown are the separate XY, XZ and YZ views of the three markers, GFP, Recoverin and Otx2 from confocal stack. (B) Shows a representative section of another example of an IRBP-GFP+ stained for GFP and rhodopsin (red) along with the XY, XZ and YZ views. Nuclei stained with DAPI in blue.