Effective Transplantation of Photoreceptor Precursor Cells Selected via Cell Surface Antigen Expression

Abstract

Retinal degenerative diseases are a major cause of untreatable blindness. Stem cell therapy to replace lost photoreceptors represents a feasible future treatment. We previously demonstrated that postmitotic photoreceptor precursors expressing an NrlGFP transgene integrate into the diseased retina and restore some light sensitivity. As genetic modification of precursor cells derived from stem cell cultures is not desirable for therapy, we have tested cell selection strategies using fluorochrome-conjugated antibodies recognizing cell surface antigens to sort photoreceptor precursors. Microarray analysis of postnatal NrlGFP-expressing precursors identified four candidate genes encoding cell surface antigens (Nt5e, Prom1, Podxl, and Cd24a). To test the feasibility of using donor cells isolated using cell surface markers for retinal therapy, cells selected from developing retinae by fluorescence-activated cell sorting based on Cd24a expression (using CD24 antibody) and/or Nt5e expression (using CD73 antibody) were transplanted into the wild-type or Crb1 rd8/rd8 or Prph2 rd2/rd2 mouse eye. The CD73/CD24-sorted cells migrated into the outer nuclear layer, acquired the morphology of mature photoreceptors and expressed outer segment markers. They showed an 18-fold higher integration efficiency than that of unsorted cells and 2.3-fold higher than cells sorted based on a single genetic marker, NrlGFP, expression. These proof-of-principle studies show that transplantation competent photoreceptor precursor cells can be efficiently isolated from a heterogeneous mix of cells using cell surface antigens without loss of viability for the purpose of retinal stem cell therapy. Refinement of the selection of donor photoreceptor precursor cells can increase the number of integrated photoreceptor cells, which is a prerequisite for the restoration of sight. STEM

Migration, Integration and Maturation of Photoreceptor Precursors Following Transplantation in the Mouse Retina

“…These new photoreceptors form synaptic connections with inner retinal neurons, generate outer segments, and are capable of restoring rod-mediated vision in models of retinal degeneration [3][4][5][6]. Importantly, we were able to show that these results are only achieved using postmitotic photoreceptor precursor cells but not progenitor cells or photoreceptors at other stages of development [1,3,7,8], a finding supported by others [9,10].…”

Defining the Integration Capacity of Embryonic Stem Cell-Derived Photoreceptor Precursors

“…However, the most efficient method for the transplantation of freshly dissociated photoreceptors is by FAC-sorting the relevant Nrl þ cell population prior to transplantation. This is also likely to be the case for ESC-derived retinal cells and the future selection of photoreceptor precursors by reporter expression or surface marker combinations may enable the improved quantification and isolation of this population within the culture [41].…”

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

“…Enrichment of rod precursor cells from the developing retina can be achieved through magnetic or fluorescent-associated cell sorting for the cell surface marker cluster of differentiation 73 (CD73). 127,128 Selection based on 2 surface markers (CD73 and CD24) increased the integration efficiency of transplanted cells 18-fold compared to unsorted cells. 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.…”

“…127,128 Selection based on 2 surface markers (CD73 and CD24) increased the integration efficiency of transplanted cells 18-fold compared to unsorted cells. 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.…”

“…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 …”