Transplantation of Photoreceptor Precursors Isolated via a Cell Surface Biomarker Panel from Embryonic Stem Cell-Derived Self-Forming Retina

Abstract

Loss of photoreceptors due to retinal degeneration is a major cause of untreatable blindness. Cell replacement therapy, using pluripotent stem cell‐derived photoreceptor cells, may be a feasible future treatment. Achieving safe and effective cell replacement is critically dependent on the stringent selection and purification of optimal cells for transplantation. Previously, we demonstrated effective transplantation of post‐mitotic photoreceptor precursor cells labelled by fluorescent reporter genes. As genetically labelled cells are not desirable for therapy, here we developed a surface biomarker cell selection strategy for application to complex pluripotent stem cell differentiation cultures. We show that a five cell surface biomarker panel CD73(+)CD24(+)CD133(+)CD47(+)CD15(−) facilitates the isolation of photoreceptor precursors from three‐dimensional self‐forming retina differentiated from mouse embryonic stem cells. Importantly, stem cell‐derived cells isolated using the biomarker panel successfully integrate and mature into new rod photoreceptors in the adult mouse retinae after subretinal transplantation. Conversely, unsorted or negatively selected cells do not give rise to newly integrated rods after transplantation. The biomarker panel also removes detrimental proliferating cells prior to transplantation. Notably, we demonstrate how expression of the biomarker panel is conserved in the human retina and propose that a similar selection strategy will facilitate isolation of human transplantation‐competent cells for therapeutic application. Stem Cells 2015;33:2469—2482

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

“…Negative selection for CD15 allows removal of contaminating proliferating progenitor cells. 129,130 These findings demonstrate that panels of surface markers are useful tools for the purification of transplantation-competent photoreceptors. The cell surface markers used to sort murine rod precursors show similar expression patterns in the developing human retina and so could help to enhance the photoreceptor yield from the iPSC retinal differentiations, as it did with the mESC.…”

“…128 This approach was recently extended to a panel of 5 cell surface markers that were used to sort rod precursor cells from mESC-3D retinal differentiation cultures. 129 At day 27 of differentiation, mESC were sorted for CD73+/CD24+/CD133+/CD47+/CD15-and transplanted in the adult murine retina; they were then able to integrate, acquire rod morphology, and form outer segments. 129 This unique combination of markers enriches for CRX+/Recoverin+/RHO+ cells transplantation-competent cells.…”

“…129 At day 27 of differentiation, mESC were sorted for CD73+/CD24+/CD133+/CD47+/CD15-and transplanted in the adult murine retina; they were then able to integrate, acquire rod morphology, and form outer segments. 129 This unique combination of markers enriches for CRX+/Recoverin+/RHO+ cells transplantation-competent cells. Negative selection for CD15 allows removal of contaminating proliferating progenitor cells.…”

“…The cell surface markers used to sort murine rod precursors show similar expression patterns in the developing human retina and so could help to enhance the photoreceptor yield from the iPSC retinal differentiations, as it did with the mESC. 128,129 Human iPSC-derived photoreceptors are reported to express CD73 at day 42 of differentiation, 122 and based on transcriptomic analysis, other potentially useful surface markers such as Cacna2d4, Kcnv2, and Cnga1 have been identified. 131 …”

Multipronged Approach to Study Glaucoma-Associated Phenotypes

“…Similarly, Feodorova et al, used size based inclusion method to isolate rod perikarya from adult mice, based on their high backscatter of heterochromatin in their nucleus (109). Lakowaski et al, used combination of five cell surface markers to facilitate the isolation of photoreceptor precursors, with a phenotype CD73(+)CD24(+)CD133(+)CD47(+)CD15(−) from three-dimensional model of retinal differentiation from mouse embryonic stem cells (110). Apart from these, flow cytometry and FACS can be utilized in many other domains and new applications have been developed in ophthalmology, for investigating ocular surface biology and pathophysiology.…”