RPE dysfunction and loss are a pivotal pathologic changes in AMD.
4,6,47,48 Because RPE cells in vivo lack the ability to functionally regenerate, cell replacement may be the only efficient way of restoring lost RPE. The RPE derived from ES cells is a potentially good cell resource for cell replacement therapy. In this study, we successfully differentiated hES cells into RPE using a two-step induction procedure. During the first step, we induced differentiation of human embryonic bodies (EBs) into neural precursors with DKK1 and noggin. DKK1, encoded by the
DICKKOPF1 gene, is a Wnt antagonist and can promote the neural differentiation of mouse EBs as evidenced by inducing the expression of the neural markers such as nestin, β-III tubulin, and distal-less homeobox gene (
DLX2).
49 –51 Noggin, a BMP signaling antagonist, can induce dorsal development and promote head formation in
Xenopus and mammals.
52 –54 Many studies have shown that noggin alone or DKK1 with lefty (a nodal antagonist) are enough to induce the differentiation of mouse or human EBs into neural precursors.
55 –57 Others have shown that endogenous level of noggin and DKK1 increase in hES cultures shortly after onset of retinal neural differentiation
58 and that treatment of hES cells with noggin, DKK1, and IGF-1 directs cells toward an anterior neural fate.
59 Therefore, we decided to first differentiate ES into neural precursors by introducing DKK1 and noggin into our neural differentiation medium, and then induced the formation of pigmented hexagonal RPE-like cells from these neural precursors with RPE differentiation medium.
40 We observed that, with this two-step induction procedure, the pigmented hexagonal RPE-like cells appeared as early as 4 weeks in cell culture, whereas with the traditional “spontaneous differentiation method,” the formation of RPE-like cells needed at least 8 weeks of cell culture.
38 Although the detailed mechanisms of DKK1 and noggin in the induction of RPE cells from hES cells remain to be elucidated, it is no doubt that DKK1 and noggin can accelerate the formation of RPE cells from ES cells in our two-step induction procedure. Recently, Idelson et al.
60 demonstrated that nicotinamide (NIC) promoted the differentiation of hES cells to neural and subsequently to RPE fate. When they cultured hES clusters with a serum-free medium supplemented with NIC, pigmented areas began to appear within the hES cell clusters after 4 weeks. They further revealed that in the presence of NIC, activin A, a member of TGF-β superfamily, significantly augmented pigmented areas within hESC clusters. This demonstration of another signaling pathway that can promote hES differentiation to RPE-like cells with similar differentiation efficiency as the blockage of Wnt and BMP pathways that we showed in this study, suggests that multiple pathways are involved in RPE differentiation in vitro.