Stem Cells – University of Washington

Can human embryonic stem cells be used to generate new retinal neurons for retinal repair?

Lamba DA, Karl MO, Ware CB, Reh TA. Efficient generation of retinal progenitor cells from human embryonic stem cells. Proc Natl Acad Sci U S A. 2006 Aug 22; 103(34): 12769-74

We have recently developed an efficient method for directing hES cells to the retinal progenitor identity based on the known regulatory factors that control eye development (Figure 1A). The current molecular genetic model of vertebrate embryogenesis suggests that there are several sequential induction steps. Forebrain development requires that both BMP and Wnt signaling are antagonized. Although the specific molecular signals required for eye field specification are not completely defined in any model system, insulin-like growth factor-1 (IGF-1) mRNA injections into Xenopus embryos specifically promote eye induction. Therefore, to direct the ES cells (Figure 1B) to an anterior neural fate, we treated embryoid bodies (Figure 1C) with a combination of noggin (a potent endogenous inhibitor of the BMP pathway) and Dickkopf-1 (dkk1; a secreted antagonist of the Wnt/beta-catenin signaling pathway) and IGF-1. The embryoid bodies are cultured for 3 days in the three factors (Figure 1A) and then transferred to 6 well plates coated with either Matrigel or laminin where they are allowed to attach (Figure 1D). The cells are then maintained in the same medium, with bFGF added, for an additional three weeks; we refer to this protocol as retinal determination (RD) conditions. Analysis of the cells with RT-PCR and immunofluorescence demonstrates that they express the full complement of eye field transcription factors, including Rx, Pax6, Lhx2, and Six3. 82% (SD+/-23%) of the cells were labeled with Pax6 antibodies at the end of 3 weeks in RD conditions (Figure 1E). Of these, 86% cells co-expressed Chx10 (SD+/-14%). Most of the Pax6 labeled cells were also labeled with antibodies to Sox2. To determine whether the hES cell derived progenitors have the capacity for multi-lineage differentiation characteristic of retinal progenitors, we used immunofluorescence for specific types of retinal neurons, including HuC/D, Neurofilament-M (Figure 2A), and Tuj-1(Figure 2B) for ganglion and amacrine cells, Crx (Figure 2D), Nrl (Figure 2C), recoverin, S-opsin, and rhodopsin for photoreceptors, and Prox1 for amacrine and horizontal cells. Many cells in the hES cultures label with markers of ganglion and amacrine cells: Tuj-1 and Neurofilament-M (while other cells are labeled with photoreceptor-specific antibodies, including Crx, and Nrl. Quantitative analysis shows on average that 12% of all cells expressed Crx (SD+/-2.4) and 12% of all cells expressed Hu C/D (SD+/-6.7) and 5.75% (SD+/-4.2) of the cells expressed Nrl (1646 cells counted). S-opsin and rhodopsin were expressed in a very small percentage of cells (<0.01%). Cells with neuronal morphology displayed synaptophysin labeled puncta, consistent with synaptic development in vitro (Figure 2E).

To analyze the functional maturation of the retinal neurons produced in these cultures, we re-dissociated the cells and plated them at lower density. This allowed us to analyze small clusters of cells with calcium imaging techniques. We found that some of the cells, particularly those with distinct neuron-like morphology, respond to glutamate, and NMDA with substantial calcium fluxes (arrow, Figure 3). Since most inner retinal neurons have glutamate receptors, and retinal ganglion cells express NMDA receptors, these data lend further support to the immunofluorescent identification of ganglion cells and amacrine cells.

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Stem Cells - University of Washington