Modeling early retinal development with human embryonic and induced pluripotent stem cells Jason S. Meyera, Rebecca L. Shearera, Elizabeth E. Capowskia, Lynda S. Wrighta, Kyle A. Wallacea, Erin L. McMillana, Su-Chun Zhanga,b, and David M. Gamma,c,d,1 aStem Cell Research Program, Waisman Center, bDepartments of Anatomy and Neurology, cDepartment of Ophthalmology and Visual Sciences, and dEye Research Institute, 1500 Highland Avenue, University of Wisconsin-Madison, Madison WI 53705 Edited by James Thomson, University of Wisconsin, Madison, WI, and approved July 23, 2009 (received for review May 15, 2009) Human pluripotent stem cells have the potential to provide compre- and maturation follows a sequence and time course highly hensive model systems for the earliest stages of human ontogenesis. reminiscent of normal retinal development. Furthermore, the To serve in this capacity, these cells must undergo a targeted, process of retinal differentiation could be selectively altered via stepwise differentiation process that follows a normal developmen- manipulation of endogenous developmental signaling pathways. tal timeline. Here we demonstrate the ability of both human embry- We then investigated whether the same culture method was onic stem cells (hESCs) and induced pluripotent stem (iPS) cells to meet capable of generating an identical cohort of developing retinal these requirements for human retinogenesis. Upon differentiation, cell types from human induced pluripotent stem cells, a recently hESCs initially yielded a highly enriched population of early eye field described source of pluripotent stem cells derived from skin cells. Thereafter, a subset of cells acquired features of advancing fibroblasts (10, 11). Cell populations expressing morphologic retinal differentiation in a sequence and time course that mimicked in features and/or markers of the eye field, retinal pigment epithe- vivo human retinal development. Application of this culture method lium, neural retinal progenitors, photoreceptor precursors, and to a human iPS cell line also generated retina-specific cell types at photoreceptors were observed in differentiating human iPS cell comparable times in vitro. Lastly, altering endogenous signaling cultures at time points predicted by results using hESCs. These during differentiation affected lineage-specific gene expression in a findings support a role for human pluripotent stem cells as in manner consistent with established mechanisms of early neural and vitro model systems to investigate mechanisms involved in retinal retinal cell fate determination. These findings should aid in the specification and differentiation of individual retinal cell types. investigation of the molecular events governing retinal specification from human pluripotent stem cells. Results Eye Field Specification from Human Embryonic Stem Cells. The appear- he study of human development is limited by a lack of model ance of eye field cells within primitive anterior neuroepithelium Tsystems that can reproduce the precise sequence and timing of is the first phase in the stepwise production of a retinal pheno- cellular and molecular events that occur during human embryo- type from an undifferentiated pluripotent stem cell (12, 13) (Fig. genesis, organogenesis, and tissue differentiation. However, the 1A). Previous reports have demonstrated that hESC-derived advent of human pluripotent stem cell technology affords a unique neuroectodermal cells will adopt anterior neuroepithelial char- opportunity to follow the full course of lineage-specific cell pro- acteristics in the absence of exogenous signaling molecules (14, duction in vitro (1, 2). The retina provides an optimal system to 15). In the current study (Fig. 1B), hESCs were differentiated as investigate this potential due to its well-defined and conserved free-floating hESC aggregates and prompted to adhere to developmental program and the availability of markers to distin- laminin-coated culture dishes to permit neural rosette forma- guish each major stage of early retinogenesis. In addition, human tion. After 16 days of differentiation, rosette-containing colonies embryonic stem cells (hESCs) display a propensity to produce cells were mechanically removed to grow as neurospheres. During this with retinal characteristics (3, 4). One criterion for assessing hESC- process, hESCs rapidly lost expression of the pluripotency genes based developmental model systems is the capacity to recapitulate Oct4 and Nanog and acquired expression of transcription factors the normal maturation sequence present in the embryo in a associated with eye field specification (Rx, Six3, Six6, Lhx2, Tll), controlled, stepwise fashion (1, 2). Preferably, such systems should anterior neural specification (Otx2) and general neural induction also provide the opportunity to test the effects of developmental (Pax6, Sox1, Sox2) (Fig. 1C). In RT-PCR experiments, Pax6 was stimuli and enrich for early cell populations, thereby reducing present as a doublet band, reflecting the expression of both the contamination from undesired and/or unidentified cell lineages. Pax6(Ϫ5a) and Pax6(ϩ5a) isoforms. The appropriate staging To date, hESC studies have focused on the derivation of and lineage of this early cell population was further supported by subsets of retinal cell populations, with emphasis on the pro- the absence of the photoreceptor precursor-specific transcrip- duction of either retinal progenitors (5, 6) or more mature cells tion factor Crx and the spinal cord-associated transcription such as retinal pigment epithelium (RPE) (3, 4) or photorecep- factor HoxB4, as well as markers of other germ layers such as tors (7). Many of these studies used various exogenous factors to increase the percentage of early retinal cell types present within brachyury (mesoderm) and alpha-fetoprotein (endoderm). Im- the mixed population of differentiating hESCs. However, despite munocytochemistry showed that nearly all cells within these these recent advances, the ability of hESCs to produce a highly enriched population of cells at the earliest stage of retinal Author contributions: J.S.M., E.E.C., and D.M.G. designed research; J.S.M., R.L.S., K.A.W., specification that can progress through each of the key devel- and E.L.M. performed research; S.-C.Z. contributed new reagents/analytic tools; J.S.M., opmental stages of the retina has yet to be demonstrated. R.L.S., E.E.C., L.S.W., K.A.W., S.-C.Z., and D.M.G. analyzed data; and J.S.M., L.S.W., and Moreover, the timing of onset of selected stages in retinal D.M.G. wrote the paper. development has varied widely among published human pluri- The authors declare no conflict of interest. potent stem cell differentiation protocols, none of which ap- This article is a PNAS Direct Submission. proximated the timeline of normal human retinogenesis (5–9). See Commentary on page 16543. We addressed these issues first by examining each major step 1To whom correspondence should be addressed. E-mail: [email protected]. in the development of definitive retinal cell populations from This article contains supporting information online at www.pnas.org/cgi/content/full/ hESCs. In doing so, we demonstrated that cell fate specification 0905245106/DCSupplemental. 16698–16703 ͉ PNAS ͉ September 29, 2009 ͉ vol. 106 ͉ no. 39 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0905245106 Downloaded by guest on September 25, 2021 A A B SEE COMMENTARY C D B E C D BIOLOGY DEVELOPMENTAL E F G H Fig. 2. Highly efficient derivation of eye field phenotypes from hESCs. (A) RT-PCR F 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 coex- pression 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 endog- enous expression of the BMP and Wnt antagonists Noggin and Dkk-1. (H) qPCR Fig. 1. Commitment toward a retinal lineage occurs as a stepwise process, showing the near complete loss of Pax6 and Rx gene expression in cells treated with beginning with the establishment of the eye field within the anterior neuroep- BMP4 and Wnt3A. (Scale bar, 40 ␮m.) ithelium. (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 The onset of Pax6 and Rx expression was detected by day 6, when differentiation. (D–F) Immunocytochemistry of typical hESC aggregates 10 days approximately 25% of all cells expressed these factors. Expression after differentiation, demonstrating the expression of the anterior neural tran- of Pax6 and Rx surpassed 90% of cells by day 10 of differentiation scription factor Otx2 (D), the eye field transcription factor Lhx2 (E), and the and increased to greater than 95% by day 16. Conversely, protein definitive neural transcription factor Sox1 (F). (Scale bar, 200 ␮m.) expression of Oct4 decreased to an undetectable level by day 10 of differentiation. The generation of a high percentage