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. Трансплантация клеток представляет собой стратегию лечения дегенерации сетчатки, характеризующуюся потерей фоторецептора. Здесь мы описываем метод обогащения трансплантируемых фоторецепторов и их субретинального прививки взрослым мышам.

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

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...