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Derivation of midbrain dopaminergic (DA) neurons from human embryonic stem (hES) cells has been of particular interest because of the clinical potential for DA neuron transplantation in patients with Parkinson's disease (PD). Several protocols for DA neuron differentiation from mouse embryonic stem cells and hES cells have been reported: however, protocols involving hES cells have yet to be improved. Here, we used a slightly modified stromal cell-derived inducing activity method, consisting four different culture stages, to show that KhES-1 cells differentiate into tyrosine hydroxylase (TH)-positive DA neurons. Quantitative real-time PCR analysis showed a marked induction of the DA neuron marker genes NURR1, paired-like homeodomain transcription factor 3 (PITX3), LIM homeobox transcription- factor 1, beta (LMX1B), engrailed-1 (EN1), dopamine transporter (DAT), and aromatic amino acid decarboxylase (AADC) during differentiation. Treatment with fibroblast growth factor (FGF)-20 and FGF-2 at the final differentiation stage induced the increase of DA neuron development-related transcription factors such as NURR1, PITX3, LMX1B, and EN1. FGF-20 and FGF-2 enhanced DA neuron differentiation from hES cell-derived neural progenitor cells directly without any soluble factors from PA6 cells. These results provide valuable information that will assist in efficient DA neuron differentiation from hES cells and for future transplant application.

Embryonic stem (ES) cells from many organisms have the capacity to generate in vitro a wide variety of cell types depending on their environment. Understanding precisely how such toti- or pluripotent cells may be driven towards a specific lineage represents a major challenge if our ambition of using ES cells to generate a ready supply of healthy cells for cell-based therapies for a range of diseases is to be realized. Recent advances have demonstrated that melanocytes and retinal pigmented epithelial (RPE) cells exhibiting the characteristics of their natural counterparts can be induced from undifferentiated ES cells grown on monolayers of specific stromal cell lines or by using a combination of Wnt3a, Endothelin-3 and SCF. The ability to induce pigment cells from ES cells promises to facilitate our understanding of the precise molecular mechanisms underlying this process and moreover enable us to distinguish the program of gene expression that underpins the choice made between generating a nerual crest-type melanocyte versus an RPE cell. Moreover, once the combination of signals required to induce a particular type of pigment cell are characterized, the way may be open for future cell-based therapy for various diseases caused by defective pigment cells.

The development of melanocytes from neural crest-derived precursor cells depends on signaling by the receptor tyrosine kinase KIT and the G protein-coupled endothelin receptor B (EDNRB) pathways. Loss-of-function mutations in either of these two signaling receptor molecules cause a loss or a marked reduction in the number of melanocyte precursors in the embryo and finally lead to loss of the coat color. Using cultures of embryonic stem (ES) cells to induce melanocyte differentiation in vitro, we investigated the requirement for EDNRB signaling during the entire developmental process of the melanocyte, in association with that for KIT signaling. During the 21-day period necessary for the induction of mature melanocytes from undifferentiated ES cells, endothelin 3 (EDN3), a ligand for EDNRB, increased the number of melanocytes in proportion to the period during which it was present. We tested the compensatory effect of EDNRB signaling on KIT signaling in vivo by using Kit(W-LacZ)/Kit(W-LacZ) ES cells and confirmed that the ectopic expression of EDN3 in the skin reduced the white spotting of Kit(W57)/Kit(W57)mice. KIT ligand (KITL) and EDN3 worked synergistically to induce melanocyte differentiation in vitro; however, the complete lack of EDNRB signaling attained by the use of EDN3-/- ES cells and an EDNRB antagonist, BQ788, revealed that the resulting failure of melanocyte development was not compensated by the further activation of KIT signaling by adding KITL. Simultaneous blockade of EDNRB and KIT signalings eliminated melanocyte precursors completely, suggesting that the maintenance or survival of early melanocyte precursors at least required the existence of either EDNRB or KIT signalings., (Copyright 2005 Wiley-Liss, Inc)

The integration of embryonic stem (ES) cells as well-differentiated neuronal cells into the retinas of adult mice was investigated. ES cells were transplanted in adult mouse retinas by intravitreal injections. Neuronal differentiation of the ES cells was investigated by morphological and immunohistochemical examinations on post-operative days 5 and 30. ES cell apoptosis was examined by in situ terminal dUTP-biotin nick end labeling of DNA fragments. Differentiated ES cells growing along the retinal surface developed fine neuronal cell processes around cell nuclei and generated neuronal networks into the retinal inner plexiform layer (IPL) 30 days after transplantation. The differentiated ES cells expressed retinal and neuronal markers. Many apoptotic cells were recognized in transplanted ES cells at day 5 but not at day 30 after transplantation. ES cells may be useful for neural tissue regeneration in the adult mammalian retina.