Cluster 6 had two subsets of genes (
Fig. 8). One group included genes that had higher expression at 3 weeks of retinal determination but that declined by 9 weeks (102 genes or ∼0.5%). This cluster included some early undifferentiated, pluripotency markers such as
POU5F1, DPPA4, LIN28, and
LIN28B. Also represented in this subcluster are
OLIG3, FEZF1, DLX5, WNT8B, WNT7B, and
PAX3. These genes are all expressed in the embryonic thalamus, near the zona limitans intrathalamica.
16 This region of the developing diencephalon appears to be induced by the RD factors, particularly at 3 weeks of treatment. Other genes in this subcluster may reflect the pattern of gene expression in this region of the CNS as well, though these have not been characterized at these stages of brain development. Alternatively, these other genes may represent a small level of contaminating cells of an unknown phenotype. Nevertheless, by 9 weeks, these genes are all at levels close to those of the fetal retina, and so it appears that this region of the CNS is not maintained through our culture conditions and purification steps. The second subset contains 115 (∼0.5%) genes that are expressed more highly in both stages of hESC cultures than in the fetal retina samples (
Fig. 8). Some of these genes are known to be expressed in ventral thalamus (SEMA3C; SLIT3),
17 RPE (PRDM16, BMP7, KRT19),
11 and ciliary epithelium (MSX2, FST),
18,19 though one of the most highly expressed genes in this subcluster, GPC3, is expressed during development in the Rathke's pouch, and the posterior wall of the diencephalon at high levels,
20 suggesting that this region of the CNS may also be induced in the hESCs by the RD conditions. Nevertheless, most of the genes in this cluster decline in expression between 3 and 9 weeks, consistent with the increasing purity of the cultures for retinal cells with our protocol.