Rapid and Efficient Directed Differentiation of Human Pluripotent Stem Cells Into Retinal Pigmented Epithelium

Controlling the differentiation of human pluripotent stem cells is the goal of many laboratories, both to study normal human development and to generate cells for transplantation. One important cell type under investigation is the retinal pigmented epithelium (RPE). Age‐related macular degeneration (AMD), the leading cause of blindness in the Western world, is caused by dysfunction and death of the RPE. Currently, RPE derived from human embryonic stem cells are in clinical trials for the treatment of AMD. Although protocols to generate RPE from human pluripotent stem cells have become more efficient since the first report in 2004, they are still time‐consuming and relatively inefficient. We have found that the addition of defined factors at specific times leads to conversion of approximately 80% of the cells to an RPE phenotype in only 14 days. This protocol should be useful for rapidly generating RPE for transplantation as well as for studying RPE development in vitro.

[1]  J. Thomson,et al.  Embryonic stem cell lines derived from human blastocysts. , 1998, Science.

[2]  G. Salvesen,et al.  Nicotinamide Rescues Human Embryonic Stem Cell‐Derived Neuroectoderm from Parthanatic Cell Death , 2009, Stem cells.

[3]  Jason Hipp,et al.  Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics. , 2004, Cloning and stem cells.

[4]  R. Pedersen,et al.  Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells , 2005, Journal of Cell Science.

[5]  D. Clegg,et al.  Protective Effects of Human iPS-Derived Retinal Pigment Epithelium Cell Transplantation in the Retinal Dystrophic Rat , 2009, PloS one.

[6]  Giuseppina Barsacchi,et al.  Specification of the vertebrate eye by a network of eye field transcription factors , 2003, Development.

[7]  B. Patel,et al.  Identification of Leptospira biflexa by real-time homogeneous detection of rapid cycle PCR product. , 1999, Journal of microbiological methods.

[8]  C. Ware,et al.  Efficient generation of retinal progenitor cells from human embryonic stem cells , 2006, Proceedings of the National Academy of Sciences.

[9]  Y. Tai,et al.  A Novel Approach for Subretinal Implantation of Ultrathin Substrates Containing Stem Cell-Derived Retinal Pigment Epithelium Monolayer , 2012, Ophthalmic Research.

[10]  L. da Cruz,et al.  Long-term outcomes following full macular translocation surgery in neovascular age-related macular degeneration , 2010, British Journal of Ophthalmology.

[11]  Ana D. Lopez,et al.  Activin A Maintains Pluripotency of Human Embryonic Stem Cells in the Absence of Feeder Layers , 2005, Stem cells.

[12]  Yoshiki Sasai,et al.  Lonely death dance of human pluripotent stem cells: ROCKing between metastable cell states. , 2011, Trends in cell biology.

[13]  D. Clegg,et al.  Pluripotent human stem cells for the treatment of retinal disease , 2012, Journal of cellular physiology.

[14]  Xuan Yuan,et al.  Activin A Maintains Self‐Renewal and Regulates Fibroblast Growth Factor, Wnt, and Bone Morphogenic Protein Pathways in Human Embryonic Stem Cells , 2006, Stem cells.

[15]  T. Adachi,et al.  Self-organizing optic-cup morphogenesis in three-dimensional culture , 2011, Nature.

[16]  Don H. Anderson,et al.  Disease susceptibility of the human macula: differential gene transcription in the retinal pigmented epithelium/choroid. , 2007, Experimental eye research.

[17]  Gideon Rechavi,et al.  Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells. , 2009, Cell stem cell.

[18]  Craig Meyers,et al.  Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor. , 2010, The Journal of clinical investigation.

[19]  H. Lin,et al.  Integrin alphavbeta5 participates in the binding of photoreceptor rod outer segments during phagocytosis by cultured human retinal pigment epithelium. , 1998, Investigative ophthalmology & visual science.

[20]  Don H. Anderson,et al.  The pivotal role of the complement system in aging and age-related macular degeneration: Hypothesis re-visited , 2010, Progress in retinal and eye research.

[21]  Yoshiki Sasai,et al.  Self-formation of optic cups and storable stratified neural retina from human ESCs. , 2012, Cell stem cell.

[22]  D. Clegg,et al.  EMBRYONIC STEM CELLS / INDUCED PLURIPOTENT STEM CELLS Derivation of Functional Retinal Pigmented Epithelium from Induced Pluripotent Stem Cells , 2009 .

[23]  N. Koizumi,et al.  ROCK inhibitor converts corneal endothelial cells into a phenotype capable of regenerating in vivo endothelial tissue. , 2012, The American journal of pathology.

[24]  Su-Chun Zhang,et al.  Modeling early retinal development with human embryonic and induced pluripotent stem cells , 2009, Proceedings of the National Academy of Sciences.

[25]  P. Kertes,et al.  Canadian expert consensus: optimal treatment of neovascular age-related macular degeneration. , 2012, Canadian journal of ophthalmology. Journal canadien d'ophtalmologie.

[26]  Chris Albanese,et al.  ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. , 2012, The American journal of pathology.

[27]  E. Carneiro,et al.  Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells. , 2008, Experimental cell research.

[28]  H. Ahmadieh,et al.  A new efficient protocol for directed differentiation of retinal pigmented epithelial cells from normal and retinal disease induced pluripotent stem cells. , 2012, Stem cells and development.

[29]  N. Benvenisty,et al.  Expanding the boundaries of embryonic stem cells. , 2012, Cell stem cell.

[30]  L. da Cruz,et al.  Induction of differentiation by pyruvate and DMEM in the human retinal pigment epithelium cell line ARPE-19. , 2011, Investigative ophthalmology & visual science.

[31]  S. Nishikawa,et al.  A ROCK inhibitor permits survival of dissociated human embryonic stem cells , 2007, Nature Biotechnology.

[32]  S. Koh VIP enhances the differentiation of retinal pigment epithelium in culture: from cAMP and pp60c-src to melanogenesis and development of fluid transport capacity , 2000, Progress in Retinal and Eye Research.

[33]  P. Brown,et al.  Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[34]  P. Bovolenta,et al.  Eye development: a view from the retina pigmented epithelium , 2004, BioEssays : news and reviews in molecular, cellular and developmental biology.

[35]  E. D. De Robertis,et al.  Neural and head induction by insulin-like growth factor signals. , 2001, Developmental cell.

[36]  G. Rubin,et al.  A comparison of macular translocation with patch graft in neovascular age-related macular degeneration. , 2009, Investigative ophthalmology & visual science.

[37]  S. Schwartz,et al.  Embryonic stem cell trials for macular degeneration: a preliminary report , 2012, The Lancet.

[38]  G. Rubin,et al.  Long‐term visual and microperimetry outcomes following autologous retinal pigment epithelium choroid graft for neovascular age‐related macular degeneration , 2008, Clinical & experimental ophthalmology.