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

Summary

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

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.

Introduction

Vision is one of the prime senses of humans. Impairment of this sense and blindness are one of the main reasons for disability in industrialized countries. The predominant cause for vision impairment or blindness is retinal degeneration, characterized by photoreceptor cell loss, as it can be observed in macular degeneration, retinitis pigmentosa, cone-rod dystrophy, and other conditions. To date, an effective therapy to restore lost vision is not available. In 2006 and 2008 two different labs reported, independent from each other, a successful transplantation of rod photoreceptor precursor cells into adult wild-type mice retinas^1,2. Thus, arising the possibility of photorecepto....

Protocol

Ethical use and care of animals statement:

All animal experiments were carried out in strict accordance with European Union and German laws (Tierschutzgesetz) and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All animal experiments were approved by the animal ethics committee of the TU Dresden and the Landesdirektion Dresden (approval number: 24D-9168.11-1/2008-33).

1. Before Starting Cell Dissociation and Cell Sorting

1. Label three 15 ml reaction tubes with: Wash (W), Positive fraction (+) and Negative fraction (-).

2. Retina Diss....

Results

In order to assess the ability of rod photoreceptors to integrate into the mouse retina, a mouse reporter line was used, in which GFP is driven by the neural retina leucine zipper (Nrl, Nrl-GFP) promoter¹¹. Nrl is the earliest marker of rod photoreceptors starting its expression at E12.5 throughout adulthood, allowing a specific labeling of donor rod photoreceptor cells.

PN 4 Nrl-GFP pups were decapitated and eyes were enucleated. Retinas were then isolated and dissociated using the.......

Discussion

Subretinal transplantation of photoreceptor precursor cells represents a reliable tool to achieve integration of these light-sensitive cells into host retinas in significant numbers^1,2. This might allow the establishment of a cell therapy for the treatment of retinal degenerative diseases in future⁶. The donor population of cells, currently isolated from PN 4 retinas, is a mixture of different cell types, from which only the photoreceptor precursor cells integrate after subretinal injection. By using CD73-based MAC-sort.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Papain Dissociation System	Worthington Biochemical Corporation	LK003150	supplied DNase I is not used in the method
Purified rat anti-mouse CD73, clone TY/23	BD Pharmingen	550738	Stock concentration 0.5 mg/ml
Goat Anti-Rat IgG MicroBeads	Miltenyi	130-048-501	Total volume of 2 ml
PBS	Gibco	10010-015	Used to count the total number of cells
DNase I	Sigma	D5025-150KU
HBSS	Gibco	14025050	Used for dissociation of the retinas
Trypan blue	Sigma	Fluka93595	Used to count the total number of cells
Vidisic	Dr. Mann Pharma / Andreae-Noris Zahn AG
Domitor	Pfizer	76579
Ketamine 10%	Ratiopharm	7538843
Antisedan	Pfizer	76590
Phenylephrin 2.5%-Tropicamid 0.5%	University Clinics Dresden Pharmacy
Preseparation Filters	Miltenyi	130-041-407
LS Columns	Miltenyi	130-042-401
MACS MultiStand	Miltenyi	130-042-303
QuadroMACS Separator	Miltenyi	130-090-976
Fire polish glass Pasteur pipette	Brand	74777 20	The pipette’s tips need to be fire-polished and autoclaved.
MACS 15 ml tube rack	Miltenyi	130-091-052
Cell count chamber	Carl Roth	T728.1
Sterile 15 ml tubes	Greiner Bio-One	188271
Leica M651 MSD	Leica	M651 MSD	can be used instead of Olympus SZX10
Olympus SZX10	Olympus	SZX10	can be used instead of Leica M651 MSD
Olympus inverted stereo microscope CKX41	Olympus	CKX41
Cell culture hood Thermo Scientific MSC-Advance	Thermo Scientific	51025411
1.5 ml Reaction tube	Sarstedt	727706400
2 ml Reaction tube	Sarstedt	72695
Eppendorf Centrifuge 5702	VWR (Eppendorf)	521-0733
Mouse head holder	myNeurolab	471030
BD Microlance 3 30 G 1/2 in	BD Pharmingen	304000
Hamilton microliter syringe 5 µl, 75RN	Hamilton	065-7634-01	delivered without needles
Hamilton RN special needle 34 G	Hamilton	065-207434	Blunt, 12 mm length
Vannas-Tübingen Spring Scissors - 5 mm Blades Straight	Fine Science Tools	15003-08
Dumont #7 Forceps - Titanium Biologie	Fine Science Tools	11272-40
Diamond pen	Tools-tech
15 mm x 15 mm Cover slips	Sparks	MIC3366