Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Abstract

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Oct4 Methylation-Mediated Silencing As an Epigenetic Barrier Preventing Müller Glia Dedifferentiation in a Murine Model of Retinal Injury

“…As previously reported (Eberle et al, 2014), isolated retinas were dissociated in DMEM containing 0.5% trypsin (Sigma) for 20 min at 37°C. The enzymatic reaction was inhibited by transferring the tissue to DMEM containing 10% fetal calf serum.…”

Daylight Vision Repair by Cell Transplantation

“…tg(Nrl −/− ; aGFP) retinas were isolated at different developmental stages (P1, 4, 6, and 8), magnetic‐activated cell (MAC)‐sorted using CD73 antibody (BD Pharmingen, Heidelberg, Germany), and 1 µl of MACS buffer containing 200,000 cells was injected into the subretinal space of host retinas as previously described in detail . Briefly, mice were anesthetized with an intraperitoneal injection of medetomidine hydrochloride (0.01 mg/10 g b.wt., Dormitor, Pfizer) and ketamine (0.75mg/10g body weight; Ratiopharm) and pupils were dilated with 2.5% phenylephrine/0.5% tropicamide (TU Dresden Pharmacy).…”

Zelltherapie am Augenhintergrund – gestern, heute, morgen

“…Darüber hinaus gibt es vielversprechende Ansätze zum Ersatz von Photorezeptoren im Tiermodell [49][50][51].…”