We like to thank Anand Swaroop for providing Nrl-GFP mice, Jochen Haas for technical support, and Sindy B&ouml;hme and Emely Lessmann for animal husbandry.	
	This work was supported by the Deutsche Forschungsgemeinschaft (DFG): FZT 111 - Center of Regenerative Therapies Dresden, the CRTD Seed Grant Program, the SFB 655, and the ProRetina e.V. foundation, the DIGS-BB Graduate Program Dresden, and the Funda&ccedil;&atilde;o para a Ci&ecirc;ncia e Tecnologia (SFRH/BD/60787/2009)

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
<ol>
	<li>Label three 15 ml reaction tubes with: Wash (W), Positive fraction (+) and Negative fraction (-).</li>
</ol>
2. Retina Dissociation
<ol>
	<li>Decapitate the PN 4 pups using a scissor.</li>
	<li>Rinse the head of the pups shortly in 70% ethanol followed by washing in PBS.</li>
	<li>Transfer the head to cold HBSS and enucleate the eye (repeat this step for all of the heads). To enucleate, open the eyelids and fixate the eye with curved forceps at the optic nerve region. Then pull the eye carefully out of the orbit.</li>
	<li>After enucleating all the eyes, isolate the retina: introduce a closed scissor into the optic nerve and open the blades. Peal the RPE/chorid out. Remove the lenses and blood vessels using curved forceps.</li>
	<li>Transfer the isolated retinas into a 1.5 ml reaction tube containing the papain solution (provided by the papain dissociation kit).</li>
	<li>Incubate the retinas in the papain solution for 30-60 min in a water bath or shaker (400 rpm) at 37 &#176;C.
		<ol>
			<li>While retinas incubate in the papain solution, pipette 1 ml of ovomucoid solution to a 15 ml reaction tube (label "1").</li>
			<li>Add 60 &#181;l of DNase I (10 mg/ml) + 60 &#181;l of ovomucoid solution (provided by the kit) + 520 &#181;l of EBSS to a 15 ml reaction tube (label "2").</li>
		</ol>
	</li>
	<li>After papain incubation, transfer the papain solution containing the partially digested retinas to reaction tube "2".</li>
	<li>Using a fire polished pipette, perform mechanical dissociation (10x up and down).</li>
	<li>Pipette the single cell suspension to reaction tube "1". Try to generate 2 layers by gently layering the cell suspension on top of the ovomucoid solution.</li>
	<li>Centrifuge for 5 min at&#160;300 x g (approximately&#160;1,300 rpm).</li>
	<li>Discard the supernatant and resuspend the cells in 500 &#181;l of MACS buffer.</li>
</ol>
3. Cell Sorting Using Magnetic Associated Cell Sorting (MACS)
<ol>
	<li>Add X &#956;l rat anti-CD73 antibody to the 500 &#181;l in order to achieve a final concentration of 10 &#956;g/ml.</li>
	<li>Incubate 5 min on ice.</li>
	<li>Fill the 15 ml reaction tube to 10 ml with MACS buffer and centrifuge it for 5 min at a speed of 300 x g.</li>
	<li>Remove the supernatant.</li>
	<li>Resuspend the pellet in 480 &#956;l MACS buffer and add 120 &#956;l of goat anti-rat IgG microbeads.</li>
	<li>Incubate 15 min on ice; do not agitate, shake or mix it.</li>
	<li>Fill the 15 ml reaction tube to 5 ml with MACS buffer and centrifuge it for 5 min at 300 x g.</li>
	<li>As the reaction tube is been centrifuged:
		<ol>
			<li>Adjust an LS Column into the magnetic stand.</li>
			<li>Put the preseparation filter on top of the LS Column.</li>
			<li>Hydrate the preseparation filter and the LS Column with 3 ml MACS buffer and collect the MACS buffer into the wash (W) tube.</li>
		</ol>
	</li>
	<li>Remove the supernatant.</li>
	<li>Resuspend the pellet in 500 &#956;l MACS buffer.</li>
	<li>Load the 500 &#956;l cell suspension to the filter, then add 1 ml MACS buffer to the filter and collect the negative fraction into the negative fraction (-) reaction tube.</li>
	<li>Next, add 3 x 3 ml of MACS buffer to the column to wash off the cells that are not bound to the column</li>
	<li>Once these 9 ml are through the LS Column, remove the column from the magnetic stand and install it on top of the positive fraction (+) reaction tube.</li>
	<li>Quickly load 5 ml of MACS buffer into the LS Column and put the plunger in the LS Column and press it down until the entire buffer is through the column.</li>
	<li>Centrifuge the reaction tube containing the positive fraction for 5 min at 300 x g.</li>
	<li>Remove the supernatant, resuspend in 500 &#181;l of MACS buffer and keep at 4 &#176;C.</li>
	<li>Count the total amount of cells using a common cell counting device.</li>
	<li>Prepare a cell suspension from the positive sorted fraction containing 2 x 105 cells/&#181;l in MACS buffer and keep at 4 &#176;C</li>
</ol>
4. Transplantation of MAC-sorted Photoreceptor Precursor Cells into the Mouse Retina
<ol>
	<li>Make sure to have all of the following solutions ready: 1 ml sterile PBS or HBSS, 10 &#181;l DNAse I, 1-2 ml sterile deionized H2O, aliquots of the cell suspension fractions at 4 &#176;C.</li>
	<li>Anesthetize the adult mouse (i.e.&#160;2-4 months old) with an intraperitoneal injection of medetomidine hydrochloride (0.01 mg/10 g body weight), ketamine (0.75 mg/10 g body weight). Then do a subcutaneous injection of Buprenorphine (0.05 mg/kg body weight) for pain relief.</li>
	<li>Dilate pupils with a drop of Phenylephrin 2.5%-Tropicamid 0.5%.</li>
	<li>Fix mouse in mouse head holder and place under stereo microscope.</li>
	<li>Apply a drop of Visidic gel to prevent drying of the eye.</li>
	<li>Make a small hole at the border between sclera and cornea (respectively ora serrata) using a sterile 30 G &#189; in&#160;needle.</li>
	<li>Cut a common cover slide into small (approximately&#160;5 mm x 5 mm) pieces using a diamond pen, place one of these pieces on top of the cornea, allowing direct visualization of the retina.</li>
	<li>Flush the presterilized microliter syringe several times with deionized water.</li>
	<li>Load the microliter syringe with 1 &#181;l of cell suspension, direct needle tangentially through the conjunctiva and sclera and place under visual control in the nasal half of the retina.</li>
	<li>Punch gently a hole into the retina until reaching the subretinal space, inject the cell suspension into the subretinal space.</li>
	<li>Observe the bullous detachment of retina, which should be uniform, covering approximately&#160;&#188; of the retinal space without any bleeding.</li>
	<li>Withdraw the syringe gently, the retinal hole seals automatically.</li>
	<li>Flush the microliter syringe several times with deionized water.</li>
	<li>Release mouse from head holder.</li>
	<li>Wake up mouse by injecting atipamozole hydrochloride for reversal of the medetomidine hydrochloride effect.</li>
	<li>Place mouse for wake up time in warm dark chamber (approximately 25 &#176;C).</li>
	<li>The next 2 days after transplantation, Buprenorphine (0.05 mg/kg of body weight) should be administered by subcutaneous injection in the morning and in the afternoon for pain relief.</li>
</ol>

<ol>
	<li>Bartsch, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+cells+integrate+into+the+outer+nuclear+layer+and+differentiate+into+mature+photoreceptors+after+subretinal+transplantation+into+adult+mice.">Retinal cells integrate into the outer nuclear layer and differentiate into mature photoreceptors after subretinal transplantation into adult mice.</a> Exp. Eye Res. 86, 691-700 (2008).</li><li>MacLaren, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+repair+by+transplantation+of+photoreceptor+precursors.">Retinal repair by transplantation of photoreceptor precursors.</a> Nature. 444, 203-207 (2006).</li><li>Eberle, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+segment+formation+of+transplanted+photoreceptor+precursor+cells.">Outer segment formation of transplanted photoreceptor precursor cells.</a> PLoS One. 7, (2012).</li><li>Lakowski, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone+and+rod+photoreceptor+transplantation+in+models+of+the+childhood+retinopathy+Leber+congenital+amaurosis+using+flow-sorted+Crx-positive+donor+cells.">Cone and rod photoreceptor transplantation in models of the childhood retinopathy Leber congenital amaurosis using flow-sorted Crx-positive donor cells.</a> Hum. Mol. Genet. 19, 4545-4559 (2010).</li><li>Barber, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+the+degenerate+retina+by+photoreceptor+transplantation.">Repair of the degenerate retina by photoreceptor transplantation.</a> Proc. Natl. Acad. Sci. U.S.A. 110, 354-359 (2013).</li><li>Pearson, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+vision+after+transplantation+of+photoreceptors.">Restoration of vision after transplantation of photoreceptors.</a> Nature. , (2012).</li><li>Singh, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+end-stage+retinal+degeneration+and+restoration+of+visual+function+by+photoreceptor+transplantation.">Reversal of end-stage retinal degeneration and restoration of visual function by photoreceptor transplantation.</a> Proc. Natl. Acad. Sci. U.S.A. 110, (2013).</li><li>Eberle, D., Schubert, S., Postel, K., Corbeil, D., Ader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+integration+of+transplanted+CD73-positive+photoreceptor+precursors+into+adult+mouse+retina.">Increased integration of transplanted CD73-positive photoreceptor precursors into adult mouse retina.</a> Invest. Ophthalmol. Vis. Sci. 52, 6462-6471 (2011).</li><li>Koso, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD73,+a+novel+cell+surface+antigen+that+characterizes+retinal+photoreceptor+precursor+cells.">CD73, a novel cell surface antigen that characterizes retinal photoreceptor precursor cells.</a> Invest. Ophthalmol. Vis. Sci. 50, 5411-5418 (2009).</li><li>Lakowski, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+transplantation+of+photoreceptor+precursor+cells+selected+via+cell+surface+antigen+expression.">Effective transplantation of photoreceptor precursor cells selected via cell surface antigen expression.</a> Stem Cells. 29, 1391-1404 (2011).</li><li>Akimoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+GFP+to+newborn+rods+by+Nrl+promoter+and+temporal+expression+profiling+of+flow-sorted+photoreceptors.">Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 3890-3895 (2006).</li><li>Eiraku, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organizing+optic-cup+morphogenesis+in+three-dimensional+culture.">Self-organizing optic-cup morphogenesis in three-dimensional culture.</a> Nature. 472, 51-56 (2011).</li><li>Nakano, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-formation+of+optic+cups+and+storable+stratified+neural+retina+from+human+ESCs.">Self-formation of optic cups and storable stratified neural retina from human ESCs.</a> Cell Stem Cell. 10, 771-785 (2012).</li><li>Osakada, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+generation+of+rod+and+cone+photoreceptors+from+mouse,+monkey+and+human+embryonic+stem+cells.">Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells.</a> Nat. Biotechnol. 26, 215-224 (2008).</li><li>Lee, M. Y., Lufkin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+&amp;#34;Three-step+MACS&amp;#34;:+a+novel+strategy+for+isolating+rare+cell+populations+in+the+absence+of+known+cell+surface+markers+from+complex+animal+tissue.">Development of the &amp;#34;Three-step MACS&amp;#34;: a novel strategy for isolating rare cell populations in the absence of known cell surface markers from complex animal tissue.</a> J. Biomol. Tech. 23, 69-77 (2012).</li></ol>

The authors declare&#160;no competing financial interests.

Subretinal transplantation of photoreceptor precursor cells represents a reliable tool to achieve integration of these light-sensitive cells into host retinas in significant numbers1,2. 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...

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 retinas1,2. Thus, arising the possibility of photoreceptor precursor cell transplantation also into a degenerated retina, to replace degenerated photoreceptors and restore vision. Indeed, it has been demonstrated recently, that such transplanted photoreceptor precursor cells elicit morphological criteria of mature wild-type photoreceptors, such as properly developed outer segments3, synaptic terminals in close proximity to endogenous bipolar cells and a round cell body located in the outer nuclear layer2-4, as well as the ability to integrate functionally into the host neural circuitry5-7.&#160;One of the main principles of this strategy is the use of post-natal day 4 (PN 4, PN0 is defined as day of birth) young mice retinas, resulting in a mixture of different cell types for transplantation. On the background of a future therapeutic&#160;application, this mixture has to be purified for photoreceptor precursor cells. CD73 has been described as the first cell surface marker specific for young photoreceptors in the retina8-10. Here, we demonstrate a photoreceptor precursor cell purification method based on this cell surface marker and with the use of the magnetic-associated cell sorting (MACS) technique. MACS might have advantages in comparison to fluorescent-activated cell sorting techniques, due to fast sorting times and the easier adjustment to GMP conditions. We could demonstrate a ~90% enrichment and an up to 3-fold higher integration rate when transplanting the enriched population to the subretinal space in adult wild-type retinas. Thus, MACS-based photoreceptor precursor cell enrichment and subretinal transplantation, are reliable and promising techniques for the development of a regenerative therapeutic strategy for the treatment of retinal degeneration.

<table border="0" cellpadding="0" cellspacing="0" width="585">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Papain Dissociation System</td>
			<td style="width:111px;">Worthington&#160; Biochemical Corporation</td>
			<td style="width:103px;">LK003150</td>
			<td style="width:176px;">supplied DNase I is not used in the method</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">purified rat anti-mouse CD73, clone TY/23</td>
			<td style="width:111px;">BD Pharmingen</td>
			<td style="width:103px;">550738</td>
			<td style="width:176px;">Stock concentration 0.5mg/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Goat Anti-Rat IgG MicroBeads</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-048-501</td>
			<td style="width:176px;">Total volume of 2ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">PBS</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:103px;">10010-015</td>
			<td style="width:176px;">Used to count the total number of cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">DNase I</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:103px;">D5025-150KU</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">HBSS</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:103px;">14025050</td>
			<td style="width:176px;">Used for dissociation of the retinas</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Trypan blue</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:103px;">Fluka93595</td>
			<td style="width:176px;">Used to count the total number of cells</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:195px;">Vidisic</td>
			<td style="width:111px;">Dr. Mann Pharma / Andreae-Noris Zahn AG</td>
			<td style="width:103px;">-</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Domitor</td>
			<td style="width:111px;">Pfizer</td>
			<td style="width:103px;">76579</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Ketamin 10%</td>
			<td style="width:111px;">Ratiopharm</td>
			<td style="width:103px;">7538843</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Antisedan</td>
			<td style="width:111px;">Pfizer</td>
			<td style="width:103px;">76590</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:195px;">Phenylephrin 2.5%-Tropicamid 0.5%</td>
			<td style="width:111px;">University clinics Dresden pharmacy</td>
			<td style="width:103px;">-</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;"></td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Name of Equipment</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td style="width:176px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Pre-Separation Filters</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-041-407</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">LS Columns</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-042-401</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">MACS MultiStand</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-042-303</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">QuadroMACS Separator</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-090-976</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">fire polish glass pasteur pipette</td>
			<td style="width:111px;">Brand</td>
			<td style="width:103px;">74777 20</td>
			<td style="width:176px;">The pipette&#8217;s tips need to be fire-polished and autoclaved.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">MACS 15ml tube rack</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-091-052</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Cell count chamber</td>
			<td style="width:111px;">Carl Roth</td>
			<td style="width:103px;">T728.1</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Sterile 15ml tubes</td>
			<td style="width:111px;">Greiner Bio-one</td>
			<td style="width:103px;">188271</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Leica M651 MSD</td>
			<td style="width:111px;">Leica</td>
			<td style="width:103px;">M651 MSD</td>
			<td style="width:176px;">can be used instead of Olympus SZX10</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Olympus SZX10</td>
			<td style="width:111px;">Olympus</td>
			<td style="width:103px;">SZX10</td>
			<td style="width:176px;">can be used instead of Leica M651 MSD</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Olympus inverted stereo microscope CKX41</td>
			<td style="width:111px;">Olympus</td>
			<td style="width:103px;">CKX41</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Cell culture hood Thermo Scientific MSC-Advance</td>
			<td style="width:111px;">Thermo scientific</td>
			<td style="width:103px;">51025411</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">1.5ml reaction tube</td>
			<td style="width:111px;">Sarstedt</td>
			<td style="width:103px;">727706400</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">2ml reaction tube</td>
			<td style="width:111px;">Sarstedt</td>
			<td style="width:103px;">72695</td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Eppendorf Centrifuge 5702</td>
			<td style="width:111px;">VWR (Eppendorf)</td>
			<td style="width:103px;">521-0733</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Mouse head holder</td>
			<td style="width:111px;">myNeurolab</td>
			<td style="width:103px;">471030</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
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subretinal transplantation of macs purified photoreceptor precursor cells into the adult mouse retina

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.

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...

Watch this Scientific Journal Video about Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina at JoVE.com

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

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.

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 ...

subretinal-transplantation-macs-purified-photoreceptor-precursor

Research

JoVE Journal

Medicine

10.6K Views.  Technische Universität Dresden. 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.