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. 2009 Sep 29;106(39):16698-703.
doi: 10.1073/pnas.0905245106. Epub 2009 Aug 25.

Modeling Early Retinal Development With Human Embryonic and Induced Pluripotent Stem Cells

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Free PMC article

Modeling Early Retinal Development With Human Embryonic and Induced Pluripotent Stem Cells

Jason S Meyer et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Human pluripotent stem cells have the potential to provide comprehensive model systems for the earliest stages of human ontogenesis. To serve in this capacity, these cells must undergo a targeted, stepwise differentiation process that follows a normal developmental timeline. Here we demonstrate the ability of both human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells to meet these requirements for human retinogenesis. Upon differentiation, hESCs initially yielded a highly enriched population of early eye field cells. Thereafter, a subset of cells acquired features of advancing retinal differentiation in a sequence and time course that mimicked in vivo human retinal development. Application of this culture method to a human iPS cell line also generated retina-specific cell types at comparable times in vitro. Lastly, altering endogenous signaling during differentiation affected lineage-specific gene expression in a manner consistent with established mechanisms of early neural and retinal cell fate determination. These findings should aid in the investigation of the molecular events governing retinal specification from human pluripotent stem cells.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Commitment toward a retinal lineage occurs as a stepwise process, beginning with the establishment of the eye field within the anterior neuroepithelium. (A) Each major stage in retinogenesis can be distinguished in part by the expression of various transcription factors. (B) Schematic of the differentiation protocol used to generate cells of a retinal lineage. (C) RT-PCR analysis of the changes in gene expression toward an eye field fate through the first 16 days of differentiation. (D–F) Immunocytochemistry of typical hESC aggregates 10 days after differentiation, demonstrating the expression of the anterior neural transcription factor Otx2 (D), the eye field transcription factor Lhx2 (E), and the definitive neural transcription factor Sox1 (F). (Scale bar, 200 μm.)
Fig. 2.
Fig. 2.
Highly efficient derivation of eye field phenotypes from hESCs. (A) RT-PCR analysis showing the onset of Pax6 and Rx gene expression and concomitant loss of Oct4. (B and C) qPCR analysis of Oct4 gene expression (B) and Pax6 and Rx gene expression (C). Values were expressed as fold change relative to undifferentiated hESCs. (D) Immunocytochemical analysis of cells at day 10 showing uniform coexpression of Pax6 and Rx (merged image includes ToPro-3 nuclear stain). (E) FACS analysis confirming the rapid loss of Oct4 expression and the onset of both Pax6 and Rx protein expression. Negative controls for FACS analyses are indicated by the white histograms. (F and G) qPCR (F) and Western analysis (G) demonstrating the endogenous expression of the BMP and Wnt antagonists Noggin and Dkk-1. (H) qPCR showing the near complete loss of Pax6 and Rx gene expression in cells treated with BMP4 and Wnt3A. (Scale bar, 40 μm.)
Fig. 3.
Fig. 3.
Acquisition of optic vesicle and optic cup cell phenotypes. (A) Mitf protein expression in neurospheres after 30 days of differentiation. (B) qPCR analysis of Mitf gene expression over the first 80 days of differentiation. (C–E) Immunocytochemical analyses of the time course of Mitf and Chx10 protein expression in neurospheres at 30 (C), 40 (D), or 50 (E) days of differentiation. (F) qPCR analysis of Chx10 gene expression over the first 80 days of differentiation. (G) Uniform Chx10 expression throughout a subset of neurospheres by day 40. (H) FACS analysis demonstrating the percentage of all cells expressing Chx10 at day 40. (I) Immunocytochemical analysis showed all Chx10+ cells coexpressed Pax6 at day 40. (J) Rosettes of Chx10+ cells expressed the tight junction protein ZO-1 within their core. (K) Rare Chx10+ cells coexpressed βIII tubulin at day 40. (L) qPCR demonstrating increased Mitf expression and corresponding decreased Chx10 expression in adherent cultures treated with the FGF inhibitor SU5402. qPCR values were expressed as fold change relative to cultures at day 16 (B and F) or day 10 (L) of differentiation. (Scale bars, 500 μm in panels A and G; 50 μm in panels C–E, J, and K; and 75 μm in panel I; blue stain in A and G is Hoechst nuclear dye.)
Fig. 4.
Fig. 4.
Generation of retinal pigment epithelium. (A) Photomicrograph of adherent cultures showing pigmented, hexagonal RPE-like cells. (B) Immunostaining revealing expression of Mitf within RPE-like cells, as well as the tight junction protein ZO-1. (C) FACS analysis demonstrating the percentage of all adherent cells expressing Mitf and Pax6 at day 40 of differentiation. (D) RT-PCR studies showing expression of genes associated with an RPE fate. (Scale bars, 100 μm.)
Fig. 5.
Fig. 5.
Generation of early photoreceptor phenotypes. (A) Immunocytochemical detection of cells expressing the photoreceptor-specific transcription factor Crx at 80 days of differentiation. (B and C) Expression of the photoreceptor protein recoverin (B) and the cone photoreceptor-specific protein opsin (C) among Crx-expressing cells at day 80. (D) RT-PCR demonstrated the stepwise acquisition of a cone photoreceptor fate from an eye field population. (Scale bars, 50 μm.) Blue stain in A is Hoechst nuclear dye.
Fig. 6.
Fig. 6.
Stepwise retinal specification from human iPS cells. (A) Various stages of retinal differentiation were observed, beginning with Pax6+/Rx+ eye field cells by day 10. (B–D) Mitf+ and Chx10+ cells, indicative of the optic vesicle/optic cup stages, are evident by day 40. (E) By day 80, clusters were present containing Chx10+ retinal progenitors and Crx+ photoreceptor precursor cells. (F–H) Many Crx-expressing cells expressed the photoreceptor protein recoverin (F) and the cone-specific protein opsin (G and H). (I) RT-PCR analysis demonstrating the stepwise expression of retina- and photoreceptor-associated genes in differentiating iPS cells over time. (J and K) RPE cells derived from iPS cells acquired a typical hexagonal morphology and pigmentation (J) and expressed Mitf and ZO-1 (K). (Scale bars, 50 μm.) Blue stain in B and D is Hoechst nuclear dye; blue stain in F is To-Pro-3 nuclear dye.

Comment in

  • Tales of retinogenesis told by human stem cells.
    Wang SZ. Wang SZ. Proc Natl Acad Sci U S A. 2009 Sep 29;106(39):16543-4. doi: 10.1073/pnas.0908643106. Epub 2009 Sep 22. Proc Natl Acad Sci U S A. 2009. PMID: 19805334 Free PMC article. No abstract available.

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