Efficient Stage-Specific Differentiation of Human Pluripotent Stem Cells Toward Retinal Photoreceptor Cells

Abstract

Recent successes in the stem cell field have identified some of the key chemical and biological cues which drive photoreceptor derivation from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC); however, the efficiency of this process is variable. We have designed a three-step photoreceptor differentiation protocol combining previously published methods that direct the differentiation of hESC and hiPSC toward a retinal lineage, which we further modified with additional supplements selected on the basis of reports from the eye field and retinal development. We report that hESC and hiPSC differentiating under our regimen over a 60 day period sequentially acquire markers associated with neural, retinal field, retinal pigmented epithelium and photoreceptor cells, including mature photoreceptor markers OPN1SW and RHODOPSIN with a higher efficiency than previously reported. In addition, we report the ability of hESC and hiPSC cultures to generate neural and retinal phenotypes under minimal culture conditions, which may be linked to their ability to endogenously upregulate the expression of a range of factors important for retinal cell type specification. However, cultures that were differentiated with full supplementation under our photoreceptor-induction regimen achieve this within a significantly shorter time frame and show a substantial increase in the expression of photoreceptor-specific markers in comparison to cultures differentiated under minimal conditions. Interestingly, cultures supplemented only with B27 and/or N2 displayed comparable differentiation efficiency to those under full supplementation, indicating a key role for B27 and N2 during the differentiation process. Furthermore, our data highlight an important role for Dkk1 and Noggin in enhancing the differentiation of hESC and hiPSC toward retinal progenitor cells and photoreceptor precursors during the early stages of differentiation, while suggesting that further maturation of these cells into photoreceptors may not require additional factors and can ensue under minimal culture conditions. STEM CELLS 2012;30:673-686Disclosure of potential conflicts of interest is found at the end of this article.

Revisiting the role of erythropoietin for treatment of ocular disorders

“…107 Various stem cells such as embryonic stem cells, [108][109][110] hematopoietic stem cells, [111][112][113] mesenchymal stem cells (MSCs), [114][115][116] induced pluripotent stem cells, 117,118 and EPC 119,120 have been shown to demonstrate the capability to regenerate into retinal neurons, 108,[110][111][112][113]117 retinal pigmented epithelium, 109,121,122 and improve blood vessels permeability 123 in both in vitro and in vivo studies. It is noteworthy that the transplanted cells will need to survive the harsh microenvironment before cell programming can occur for the replacement of damaged tissue.…”

Strategies for retinal cell generation from human pluripotent stem cells

“…S-opsin was expressed by ~70% of differentiated cells at this time point. The additional blocking of TGFβ signaling appears to be paramount to the success of this protocol as compared to previous methods that inhibit only BMP and Wnt or Wnt and Nodal signaling (63)(64)(65)(66)(67). These findings also suggest that antagonizing all of these signaling pathways is required for cone genesis at the expense of rod genesis, which measured by marker expression appeared to be the more prevalent photoreceptor in the retinal cultures of the previously established protocols.…”

“…Mellough et al combined some aspects of these two previous studies with additional modifications to mimic neural and retinal development in an attempt to design a photoreceptor differentiation protocol for hESC and hiPSC with higher yields and less culture time (67). The threestep differentiation resulted in 16% of CRX + cells and the appearance of late-stage photoreceptor-specific markers RHO, OPN1LW, and OPN1SW by day 45, representing a dramatic decrease in time to differentiation from previous reports (65).…”

Induced Pluripotent Stem Cell Therapies for Degenerative Disease of the Outer Retina: Disease Modeling and Cell Replacement

“…Notably, to obtain photoreceptors in vitro, the WA09 line (otherwise called H9 cells), derived at the University of Wisconsin (USA), has been often used. [28][29][30][31] These cells maintain proliferation capacity, express high levels of telomerase, and do not show karyotypic aberrations when cultured for as long as 14 months on irradiated mouse embryonic fibroblasts (MEF). 11,32,33 Nevertheless, karyotypic changes have been reported in H9 cells in the same culture conditions, including frequent changes in chromosome 12 where the gene NANOG is located.…”

“…30 To avoid the presence of animal derivatives (animal-derived sera, or in most cases Matrigel), a chemically defined 3D retinal differentiation medium was recently reported, which only contains nicotinamide and 20 ng/mL of basic fibroblast growth factor. 121 To date, efforts to speed up the differentiation protocols 31,122,123 using molecules known to promote retinal differentiation 31,118,123 or by passing the EB formation 122 have generally reduced the survival of differentiated cells, 31,123 and/or the stratification obtained. 122 Whether correct lamination of pluripotent stem cell-derived donor retinal tissue is required for generation of transplantationcompetent human photoreceptor cells remains to be resolved.…”