Increased Integration of Transplanted CD73-Positive Photoreceptor Precursors into Adult Mouse Retina

Abstract

PURPOSE. Retinal degeneration initiated by loss of photoreceptors is the prevalent cause of visual impairment and blindness in industrialized countries. Transplantation of photoreceptor cells represents a possible replacement strategy. This study determined that identification of cell surface antigens can assist in enriching photoreceptor precursors for transplantation. METHODS. The expression profile of rod photoreceptors at postnatal day 4 was investigated by microarray analysis to identify photoreceptor-specific cell surface antigens. For enrichment of transplantable photoreceptors, neonatal retinas from rod photoreceptor-specific reporter mice were dissociated, and the rods were purified by magnetic associated cell sorting (MACS) with CD73 antibodies. MAC-sorted cell fractions were transplanted into the subretinal space of adult wild-type mice. The number of rod photoreceptors contained in unsorted, CD73-negative, and CD73-positive cell fractions were quantified in vitro and after grafting in vivo. RESULTS. Microarray analysis revealed that CD73 is a marker for rod photoreceptors. CD73-based MACS resulted in enrichment of rods to 87%. Furthermore, in comparison with unsorted cell fractions, CD73-positive MAC-sorted cells showed an approximately three-fold increase in the number of integrated, outer segment-forming photoreceptors after transplantation. CONCLUSIONS. CD73-based MACS is a reliable method for the enrichment of integrating photoreceptors. Purification via cell surface markers represents a new tool for the separation of transplantable photoreceptor precursors from a heterogeneous cell population, avoiding the need of reporter gene expression in target cells.

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].…”

“…The capacity to migrate may thus be restricted even within the postmitotic precursor population. Defining whether such a window exists is of crucial importance for improving transplantation efficiency, for the identification of accurate cellsurface markers [8,10], and for the isolation of a homogeneous population of integration-competent donor cells from the otherwise heterogeneous populations that arise from stem cell differentiation protocols [11,13,16,58]. Here we found that cells that migrate into the recipient retina are universally rod a-transducin -ve , despite the presence of rod a-transducin + ve cells in the subretinal space for at least 6 weeks posttransplantation (indicating that survival of more mature cells is not an issue).…”

Reversal of end-stage retinal degeneration and restoration of visual function by photoreceptor transplantation

“…We used a severe and rapidly degenerating murine model, the rd1 mouse, in order to effectively model the anatomy in end-stage human retinal degenerations. Models used in previous studies included wild-type (3,8,9,25,27,(30)(31)(32)(33), rd9 Precursor transplantation improved pupil constriction, compared with no transplant controls (F 2,34 = 7.264, P = 0.002, n = 11-14 each; **P = 0.001 post hoc). (E) Paired analysis before versus after treatment showed that at the median light stimulus intensity, the PLR improved significantly in precursortreated animals (***t 11 = 5.95, P < 0.0001, n = 12).…”

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.…”