Date Published: January 20, 2010
Publisher: Public Library of Science
Author(s): Deepak A. Lamba, Andrew McUsic, Roli K. Hirata, Pei-Rong Wang, David Russell, Thomas A. Reh, Rafael Linden. http://doi.org/10.1371/journal.pone.0008763
Abstract: Inherited and acquired retinal degenerations are frequent causes of visual impairment and photoreceptor cell replacement therapy may restore visual function to these individuals. To provide a source of new retinal neurons for cell based therapies, we developed methods to derive retinal progenitors from human ES cells.
Partial Text: Retinal degenerations that involve rod and cone photoreceptors are a major cause of blindness, and affect millions of people in the US. These devastating conditions can be inherited or acquired, and while efforts are underway to develop treatments that slow or prevent these conditions using gene therapy or medical treatments, once the photoreceptors have degenerated, cell replacement or prosthetic devices are the only options. Cell replacement of photoreceptors has been shown to be feasible, even in mature mice, where photoreceptors transplanted to the sub-retinal space can integrate into the retina and function , . We, and others, have developed protocols for generating retinal progenitors and photoreceptors from human embryonic stem cells as a potential source of replacement photoreceptors for cell-based therapy of retinal degenerations , , . Our protocol involves the directed differentiation of undifferentiated human embryonic stem (hES) cells into retinal progenitor cells, followed by expansion of these cells and their differentiation into photoreceptors. These cells can be transplanted to the sub-retinal space of visually deficient mice and can restore some light response .
The iPS cells were generated from normal human fibroblast cultures as described previously (Yu et al., 2007) with lentiviral vectors expressing OCT4, NANOG, LIN28 and SOX2. Upon quadruple infection, several human ESC-like colonies appeared after three weeks and were isolated and expanded further. One particular clone (iPSC-MHF2 c1) was chosen for our experiments. When cultured, these cells expressed the pluripotency markers SOX2, OCT4 and SSEA-4 (figure 1A-A″′). The cells could be maintained with CF-1 feeder layers as well as in feeder-free MEF-conditioned media. In vivo, the iPS cells were capable of differentiation into cells of all three germ layers upon teratoma formation in immunodeficient mice (Figure 1B, C, D), confirming that the iPSCs were pluripotent. Additionally, we looked for silencing of exogenous pluripotency genes and found that even though Nanog and Lin28 were silenced, there was still some expression of lentivirus induced Oct4 and Sox2 (Supplemental Figure S2).
In this report we have shown that human iPS cells can be used to generate retinal photoreceptors that can be purified by infecting with a lentivirus that drives GFP from the IRBP promoter and subsequent FACS. Our results show for the first time that human photoreceptors derived from either ES cells or iPS cells can be purified using a combination of photoreceptor-specific GFP vector and fluorescent activated cell sorting. Together with previous results that demonstrated the potential for ES cell derived photoreceptors to integrate following transplantation and restore light response to CRX −/− mice , the results presented in this report further support the possibility that stem cell approaches can lead to therapies for the treatment of retinal degenerations.