Microarray Characterization of Human Embryonic Stem Cell–Derived Retinal Cultures

Hereditary and age-related photoreceptor degenerative diseases can lead to the death of sufficient numbers of photoreceptor cells to cause significant visual impairment and blindness. The mammalian retina does not have an innate capacity to regenerate the dying cells (see Ref. 1 for review); visual prosthetics or cell replacement strategies are therefore being pursued in an effort to restore some vision to the most severely affected patients. Human embryonic stem cells (hESCs), because of their property of pluripotency, may have a significant role in cell replacement therapies in the eye. However, this same property requires that the cells be directed in their differentiation to retinal cell fates before use in therapies to minimize the risks of developing teratomas. Several groups, including our own, have previously shown that a combination of signaling molecules can direct ESCs to retinal cell fates (see Discussion). The retinal determination (RD) protocol we developed uses a combination of a BMP inhibitor (e.g., Noggin), a Wnt Inhibitor (DKK-1), and the growth factor IGF-1 (RD factors). With the RD protocol we can specifically induce retinal determination in hESCs,2 hiPSCs,3 and mouse ESCs (La Torre et al., unpublished observations, 2010) with very high efficiency. We also found that photoreceptors from these hESC-derived retinal progenitors have the capacity to convey some light responsiveness in a mouse model of Leber congenital amaurosis4 showing in principle that cell-based replacement therapy might be applicable to some forms of inherited retinal degeneration. Before these hESC-derived retinal cells can be used therapeutically, they must be highly characterized. The presence of undifferentiated ESCs in a transplant may result in teratoma formation in the recipient eye, whereas the presence of other ESC-derived tissues, such as muscle or bone, would likely interfere with normal visual function. In our earlier studies, we used quantitative PCR (QPCR) to assess the levels of markers of the various lineages. However, these studies provided only a limited view of the status of differentiation of the population of cells treated with the RD factors. Here, we instead used cDNA microarrays to get a more global view of the efficiency of our RD protocol. We performed microarray analysis on hESC-derived cells from two different time points (3 weeks and 9 weeks) and on human fetal retinas from three different ages (60, 80, and 96 days after conception). This allowed us to compare the efficiency of our protocol and to stage our cells with relation to human retinal development in vivo. Our results indicate that most cells in the cultures treated with the RD protocol develop as retinal progenitors and ultimately differentiate into retinal cells. Markers for nonocular tissues were absent or expressed at very low levels, though we did find evidence for the presence of pigmented epithelial cells and ciliary epithelial cells in the cultures. This is the first study to assess the purity of hESC-derived retinal cells at the transcriptome level, and it provides a baseline for comparison for future studies using other directed differentiation protocols.