Abstract
Retinal degeneration leads to loss of light-sensing photoreceptors eventually resulting in vision impairment and impose a heavy burden on both patients and the society. Currently available treatment options are very limited and mainly palliative. Ever since the discovery of human pluripotent stem cell technologies, cell replacement therapy has become a promising therapeutic strategy for these patients and may help restore visual function. Reproducibly generating enriched retinal cells including retinal progenitors and differentiated retinal neurons such as photoreceptors using human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells in a dish is an essential first step for developing stem cell-based therapies. In addition, this will provide a reliable and sufficient supply of human retinal cells for studying the mechanisms of diseases. Here we describe a small molecule-based retinal induction protocol that has been used to generate retinal progenitors and differentiated retinal neurons including photoreceptors from several human ES and iPS cell lines. The retinal cells generated by this protocol can survive and functionally integrate into normal and diseased mouse retinas for several months following subretinal transplantation.
Keywords: Human, ES Cells, iPS Cells, Retina, Differentiation
Background
A number of groups around the world are developing methodologies to generate specific cell types from human pluripotent stem cells. These cells will likely play a critical role in the future of regenerative medicine as a source of replacement cells. These newly generated human cells will be very useful in developing better and more accurate human disease models that can then be used for discovery of novel drugs with better efficacy and safety profiles. Our work focuses on retinal degenerative diseases such as macular degeneration and retinitis pigmentosa which affect millions of people worldwide. Death of light-sensing photoreceptors in the retina is commonly associated with those diseases and results in severe impairment or total loss of vision. There are no effective medical treatments available to cure those diseases. Under specific conditions, human ES and iPS cells can be specifically used to generate retinal progenitor cells, and consequentially differentiate into specialized retinal neuronal subtypes (retinal ganglion cells, amacrine cells, bipolar cells, horizontal cells, and photoreceptors) (Osakada et al., 2008 and 2009; Hirami et al., 2009; Lamba et al., 2006, 2009 and 2010; Meyer et al., 2009; Hambright et al., 2012; Zhong et al., 2014). Establishment of effective and chemically-defined protocols to generate retinal progenitors as well as differentiated retinal cell types including photoreceptors from human ES cells and iPS cells is a critical step for developing cell replacement therapies for patients with a variety of incurable retinal degenerative diseases.Here, we report a detailed small molecule-based retinal induction protocol that has been used to generate retinal cells in vitro and the derived retinal cells were used as donor cells in the transplantation studies carried out by Dr. Lamba’s research group. The derived retinal progenitors and retinal photoreceptors were tested in multiple host mouse lines with and without retinal degeneration conditions and showed the ability to survive and functionally integrate into the host mouse retina following transplantation (Zhu et al., 2017 and 2018).
Materials and Reagents
Equipment
Procedure
Data analysis
Data analysis for the differentiated retinal cells is carried out by t-test. Total cells expressing a given antibody as well as total cells in a field (marked by DAPI) were counted from 3-5 fields and at least 3 independent experiments. The results were compared to the marker expression in either undifferentiated cells or alternate differentiation media. Detailed analysis of induced retinal cell differentiation of human ES cells and iPS cells via this protocol can be found in a recently published open access paper by the lab (Zhu et al., 2018).
Notes
While the protocol works reliably in multiple cell lines tested by our lab. There is some inherent variability due to different culture conditions experienced by various undifferentiated cells or methods of derivation. Authors also advise all labs to routinely test lines for mycoplasma contamination using commercial kits as they significantly affect differentiation capacity.
Recipes
Acknowledgments
We are grateful to Dr. Xianmin Zeng for the human iPS cell lines (NCL-1, NCL1-GFP). The human ES line was purchased from WiCell. The work was supported through funds from NEI grant EY025779, Buck Institute funds. This protocol is based on our previous work (Zhu et al., 2018).
References
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