Isolation, Concentration, and Expansion Culture of Human Rod Precursors and Extracellular Vesicles Derived Therefrom

Abstract

Methods for producing concentrated populations of precursor rod photoreceptor cells (PRP) and EVs derived therefrom, and methods of using PRP and EVs for diagnosing and treating retinal diseases are described herein.

Description

Technical Field

[0001] Claim of Priority This application claims priority to U.S. Provisional Patent Application No. 63 / 339,252, filed May 6, 2022. The entire foregoing is incorporated herein by reference.

[0002] Technical Field Methods for producing enriched populations of progenitor rod photoreceptor cells (PRP) and extracellular vesicles (EV) derived therefrom, as well as methods for using PRP and EV for diagnosing and treating retinal diseases are described herein.

Background Art

[0003] Retinal degenerative diseases that result in the death of rod and cone photoreceptor cells are the leading cause of hereditary vision loss worldwide. For example, retinitis pigmentosa is a disease in which rod photoreceptors die first, leading to the death of cone photoreceptors. Induced pluripotent stem cells or embryonic stem cells (iPSC / ESC) have been proposed as a potential source of new photoreceptors to restore vision in these conditions. Studies conducted over the past decade in mouse models of retinal degeneration have highlighted several limitations regarding cell replacement or neuroprotection in the retina, such as poor integration of transplanted cells in the host retina and the need for high drug doses to neuroprotect the retina.

Summary of the Invention

[0004] Methods for providing a population of progenitor rod photoreceptor cells (PRP) are described herein. The method can include providing an initial population comprising retinal cells from a mammal, preferably a fetal mammal (alternatively, the initial population can comprise cells derived from a retina or an eye cup organoid generated from iPSC or ESC). Cells expressing CD73 and CD276 (and optionally Cd11b) are isolated from the initial population to provide a substantially purified population of PRP.

[0005] In some embodiments, the step of isolating cells that express CD73 and CD276 (and optionally Cd11b) includes using fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS), optionally in a microfluidic device. For example, the method can include contacting an initial population of cells with antibodies that bind to CD73 and CD276, and isolating the cells to which the antibodies to both have bound.

[0006] In some embodiments, the method further includes culturing an initial population that includes retinal cells prior to isolating the cells that express CD73 and CD276 (and optionally Cd11b).

[0007] The method can optionally include culturing a substantially purified population of the obtained CD73+ / CD276+ PRP. In some embodiments, the substantially purified population of PRP is cultured in a medium that includes serum or serum replacement, EGF, and FGF. In some embodiments, the substantially purified population of PRP is cultured under hypoxic conditions (e.g., 3-10% O 2 ). In some embodiments, hPRP is also CD11b+.

[0008] In some embodiments, the initial population that includes retinal cells is from a fetal mammal, e.g., human.

[0009] Similarly, a substantially purified population of PRP produced by the methods described herein is provided herein. In some embodiments, at least 75%, such as at least 80%, 85%, 90% or 95% of the population is composed of cells that express both CD73 and CD276 (and optionally Cd11b). Further provided is a substantially purified population of PRP for use in the treatment of a subject having a condition associated with the loss of retinal rods or cones photoreceptors described herein. In some embodiments, the condition associated with the loss of retinal rods or cones photoreceptors is a hereditary retinal degenerative disease (IRD), optionally cone-rod dystrophy, retinitis pigmentosa (e.g., LCA) or Stargardt disease; or macular degeneration, e.g., atrophic macular degeneration. Similarly, provided herein is a composition comprising the PRP described herein (e.g., where at least 75%, such as at least 80%, 85%, 90% or 95% of the population is composed of cells that express both CD73 and CD276 (and optionally, Cd11b)), optionally formulated in a physiologically acceptable buffer or polymeric gel, e.g., as described herein, optionally a gelatin-hyaluronic acid (HA) hydrogel, e.g., a composition comprising gelatin hydroxyphenylpropionic acid (gelatin-HPA) and hyaluronic acid-tyramine (HA-Tyr).

[0010] Additionally, provided herein is a method of treating a subject having a condition associated with the loss of retinal rod photoreceptors. The method may include administering to the subject (e.g., by injection) a therapeutically effective amount of PRP obtained by the methods described herein, preferably where at least 75%, such as at least 80%, 85%, 90% or 95% of the population is composed of cells that express both CD73 and CD276 (and optionally Cd11b), and optionally formulated in a physiologically acceptable buffer or polymeric gel, e.g., as described herein, optionally a gelatin-hyaluronic acid (HA) hydrogel, e.g., a composition comprising gelatin hydroxyphenylpropionic acid (gelatin-HPA) and hyaluronic acid-tyramine (HA-Tyr).

[0011] Furthermore, provided herein is a method of obtaining or providing extracellular vesicles (EVs) derived from rod photoreceptor precursor cells. The method can include providing a substantially purified population of PRP as described herein; maintaining the substantially purified population of PRP in a culture medium, preferably, the culture medium is contacted with the PRP in the culture for at least 12, 18, 24, 36, 48, or 72 hours; removing the culture medium from the culture; and isolating EVs from the culture medium.

[0012] In some embodiments, the step of isolating EVs includes ultracentrifuging the culture medium to obtain a pellet containing the EVs. An exemplary ultracentrifugation protocol includes 3 steps: 1500g for 30 minutes; 10,000g for about 20 minutes; and 15,000g for 30 minutes.

[0013] In some embodiments, the method further includes lyophilizing the isolated EVs.

[0014] Similarly, provided herein are compositions comprising EVs obtained by the methods described herein. In some embodiments, the EVs are in a polymeric gel scaffold, such as a polymeric scaffold comprising polycaprolactone (PCL), polylactic-co-glycolic acid (PLGA), polyethylene glycol (PEG), polylactic acid (PLA), polyetherimide (PEI), PNIPAAM, or hydroxyethyl-methacrylate. Similarly, provided are compositions for use in treating a subject having a condition associated with loss of retinal rods or cones. Additionally, provided herein is a method of treating a subject having a condition associated with loss of retinal rods or cones as described herein. The method can include administering to the subject a therapeutically effective amount of PRP obtained by the methods described herein.

[0015] In some embodiments, conditions associated with the loss of retinal rod or cone photoreceptors are hereditary retinal degenerative diseases (IRDs), optionally, cone-rod dystrophy, retinitis pigmentosa (e.g., LCA) or Stargardt disease; or macular degeneration, e.g., atrophic macular degeneration.

[0016] As used herein, unless otherwise specified, the term "about" means ±10% of the indicated number.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials well known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database registrations, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0018] Other features and advantages of the invention will become apparent from the following detailed description, figures, and claims.

Brief Description of the Drawings

[0019]

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Mode for Carrying Out the Invention

[0020] Methods and compositions are described herein that address the limitations and drawbacks associated with previous methods in the isolation, purification, and concentration of rod progenitor cells. Methods for producing enriched populations of human progenitor rod photoreceptor cells (hPRP) and extracellular vesicles (EVs) derived therefrom, and methods of using hPRP and EVs for diagnosing and treating retinal diseases are provided.

[0021] human progenitor rod photoreceptor cells (hPRP) Methods for the isolation, purification, and expansion of rod photoreceptor progenitor cells, such as purified or enriched populations of rod photoreceptor progenitor cells, e.g., human progenitor rod photoreceptor cells (hPRP), methods for producing these cells, and the use of the cells for the treatment of eye disorders, e.g., retinal degenerative diseases and other diseases are provided herein. For example, a microfluidic-based cell sorting approach is described that includes an expandable GMP-compliant protocol for the production of human progenitor rod photoreceptors, resulting in the ability to generate universal, allogeneic rod photoreceptor cells to preserve and restore vision, as shown, for example, in FIG. 1.

[0022] As shown herein, the combination of CD73 / CD276 is useful as a surface marker for labeling and isolating rod cells. CD276 has been found to be upregulated in rod progenitor cells and can be released from the membrane and to the cell surface. The inventors used this to label rod progenitor cells (CD276 is exclusively expressed only by rod progenitor cells). CD73 is also well known as 5'-nucleotidase ecto (NT5E); exemplary sequences for CD73 are provided in GenBank under accession numbers NP_002517.1 (5'-nucleotidase isoform 1 preproprotein) and NP_001191742.1 (5'-nucleotidase isoform 2 preproprotein). Exemplary sequences for CD276 are provided in GenBank under accession numbers NP_001019907.1 (CD276 antigen isoform a precursor); NP_001316557.1 and NP_079516.1 (CD276 antigen isoform b precursor); and NP_001316558.1 (CD276 antigen isoform c).

[0023] Fetal retinal cells (e.g., at human embryonic age (fetal age) 10-16 weeks, or at a similar age in development for other mammals, e.g., mouse) are optionally under hypoxic conditions (3-10% O 2) In (0), it is cultured using a nutrient mixture optionally containing the serum described herein (e.g., fetal bovine serum (FBS)) or a serum substitute (e.g., PHYSIOLOGIX Xeno-Free Serum Replacement (Nucleus Biologics) or KNOCKOUT Serum Replacement (Thermo Fisher)), EGF, and FGF, resulting in the time frame with the highest expression of rod progenitor cells during retinogenesis; differentiated iPSCs and ESCs can also be used. When iPSCs / ESCs are used, the ESCs / iPSCs grow into eye cups or retinal organoids, and the organoids (e.g., on days 40 to 60) are used as a starting point for sorting using methods described herein, for example, based on the presence of CD73 and CD276. See, for example, Nakano et al., Cell Stem Cell. 2012 Jun 14;10(6):771-785; Kuwahara et al., Methods Mol Biol. 2017;1597:17-29; Eiraku et al., Nature. 2011 Apr 7;472(7341):51-6; Reh and Fischer, Methods Enzymol. 2006;419:52-73; Fathi et al., Front Neurosci. 2021 Apr 20;15:668857; and Achberger et al., Adv Drug Deliv Rev. 2019 Feb 1;140:33-50.

[0024] The present technology can be used for the isolation of rod progenitor cells, optionally using a microfluidic device, such as the MACSQUANT TYTO sterile cartridge microfluidic cell sorting system (Miltenyi), to provide a concentrated population of cells with a higher purity and viability than previously reported. The microfluidic device enables the capture of cells labeled with both positive markers (CD73 / CD276) and / or the removal of a population of cells that were not needed (all the remaining cells that are CD73- and / or CD276-). The method can be used to obtain a population of cells in which at least 75%, for example, at least 80%, 85%, 90% or 95% of the cells are positive (expressing) for both CD73 and CD276. In this experiment, the confirmatory marker recoverin, CRX, CD73 and CD276 expression showed approximately 95% positivity for these markers. Furthermore, maintaining the isolated cells using the culture conditions described herein enabled cell proliferation to obtain a large number of hPRP. When transplanted into the mouse eye, hPRP cells showed a very high ability to provide neuroprotection in a degeneration model. As far as the inventors know, this specification is the first description of a method for isolating and culturing highly pure rod progenitor cells that can provide high neuroprotection to the retina.

[0025] hPRP cells can be differentiated to provide a population of human rod photoreceptor cells. A number of protocols are well known; an exemplary protocol includes culturing the cells in DMEM / F12 3:1 + KSR / FBS containing 0.1% 2-ME (β-mercaptoethanol), 0.2% IGF-1 (IGF1 recombinant human protein), 2% B27 supplement, 1% taurine and 1 μM 9-cis retinal. The medium is changed approximately every 2 days and the cells mature into rod photoreceptors in approximately 60 to 90 days.

[0026] Extracellular vesicles (EVs) derived from human progenitor rod photoreceptor cells (hRP) EVs and microvesicles are cell fragments encapsulated in lipids with diameters ranging from 50 nm to 2 mm; they can be derived from almost all cell types, including embryonic stem cells, hematopoietic stem cells, neurons, and malignant cells. Typically, EVs have a diameter of 30 - 130 nm and are formed through the endosomal sorting complex required for transport (ESCRT). Microvesicles have a diameter of 100 - 2000 nm and are heterogeneous in size. While EVs are within the size range of microvesicles, microvesicles differ in formation, secretion, and content. The formation of microvesicles involves interactions between the cytoskeleton and the phospholipid proteins of the plasma membrane. EVs and microvesicles have been shown to be involved in cell - cell communication via the transfer of proteins, mRNA, miRNA, and DNA, as shown, for example, in Figure 2. EVs and microvesicles encapsulate representative factors of the original genotype and phenotype of the cells.

[0027] EVs derived from human progenitor photoreceptor cells (hPRPs) isolated using the methods described herein are provided herein. Some or all of the proteins shown in Table 1 are preferably present in the EVs for use in the methods and compositions, and optionally, thioredoxin (TXN, RDCVF; also known as NP_001231867.1); fibulin extracellular matrix protein 1-containing EGF (EFEMP1; NP_001034437.1); glutathione S-transferase Mu isoform 1 (GSTM1; NP_000552.2 or NP_666533.1); sarcoglycan delta (SGCD; NP_000328.2, NP_758447.1 or NP_001121681.1); staphylococcal nuclease and tudor domain-containing 1 (SND1; NP_055205.2), ADAM metallopeptidase domain 9 (ADAM9; NP_003807.1); and ezrin (EZR; NP_003370.2), including at least 1, 2, 3, 4, 5 or all 7 of them. Thereby, the EVs can contain specific proteins, including 1, 2, 3, 4, 5, 6 or all 7 of TXN, EFEMP1, GSTM1, SGCD, SND1, ADAM9 and EZR. Without being bound by theory, these proteins are thought to provide a therapeutic effect. An overview and figure of exemplary EVs are shown in Figure 2. Antibody-directed targeting of EVs increases the specificity of delivery of neuroprotective proteins, miRNAs and other molecular cargo in EVs to photoreceptors. This can be used to facilitate the delivery and internalization of appropriate concentrations of neuroprotective cargo by photoreceptors to activate recovery and repair processes. Methods for targeting EVs for delivery of therapeutic agents have been well demonstrated in non-ocular tissues (see, for example, Chen et al., Front. Cell Dev. Biol., 08 October 2021. doi.org / 10.3389 / fcell.2021.751079).

[0028] hPRP cells continuously secrete EVs into the culture medium, for example, throughout the manufacturing and culturing processes. EVs can be obtained from hPRP cells using well-known methods. An exemplary manufacturing method is summarized in Figure 3. In this example, EVs secreted by hPRP cells are obtained from their culture medium. The medium is frozen and then ultracentrifuged to isolate the EVs and discard debris, dead cells, and other unwanted components. The final precipitate is resuspended in PBS, lyophilized, stored, and kept the EV structure intact. Finally, the EVs are resuspended in an aqueous carrier (optionally including PBS or a hydrogel carrier) and injected into the vitreous of the eye. The medium is selected to contain the secreted EVs.

[0029] EV or hPRP can be suspended, for example, in a physiologically acceptable buffer, such as phosphate buffered saline (PBS), or in a biocompatible polymer, such as a hydrogel. EVs injected simply in phosphate buffered saline (PBS) can be targeted by the host immune system, thereby reducing their effectiveness in inducing regeneration, and polymeric scaffolds containing biocompatible biomaterials may be used to encapsulate EVs prior to delivery; hPRP can also be formulated in a polymeric gel for administration. Suitable biodegradable and biocompatible polymeric materials may include one or more of gelatin, chondroitin sulfate, hyaluronic acid, alginic acid, collagen, and chitosan-based scaffolds, such as gelatin-hyaluronic acid gels, for example, compositions containing gelatin hydroxyphenylpropionic acid (gelatin-HPA) and hyaluronic acid-tyramine (HA-Tyr) (see, for example, WO 2021 / 113515 pamphlet and Dromel et al., NPJ Regen Med. 2021 Dec 20;6(1):85). Such materials provide non-toxic and biocompatible degradation products while causing a lower immune response than synthetic polymers themselves. Other materials that can be used include, but are not limited to, polycaprolactone (PCL), polylactic-co-glycolic acid (PLGA), polyethylene glycol (PEG), polylactic acid (PLA), polyetherimide (PEI), PNIPAAM, or hydroxyethyl-methacrylate. See, for example, US Patent No. 2014 / 0234381 and US Patent No. 2011 / 0004304.

[0030] Method of Use The EVs and hPRPs produced as described herein can be used to treat retinal degenerative diseases such as those associated with the loss of photoreceptor rods or cones, such as macular degeneration, atrophic macular degeneration; retinitis pigmentosa, etc.; and other hereditary retinal diseases such as Stargardt disease, Leber congenital amaurosis (LCA) or cone-rod dystrophy (CRD); in particular, the method can be used in a state where rods are first lost. For example, certain retinal diseases affected by cone loss (e.g., secondary cone loss in retinitis pigmentosa (RP) leads to blindness) can be treated using hPRP or EVs isolated from the hPRP cultures described herein; in some embodiments, the EVs contain rod-derived cone viability factor (RdCVF), an inactive thioredoxin secreted by rod photoreceptors that protects cones from degeneration (Ait-Ali et al., Cell. 2015 May 7;161(4):817-32). Degeneration of retinal photoreceptors is also seen in the late stage of atrophic AMD and is also known as geographic atrophy (GA). In some embodiments, the method may include administering a population of hPRPs to replace cells lost in the degenerative disease. Hereditary retinal diseases that can be treated by the methods described herein include Bardet-Biedl syndrome, autosomal recessive; choroidoretinal atrophy or degeneration, autosomal dominant; cone or cone-rod dystrophy, autosomal dominant; cone or cone-rod dystrophy, autosomal recessive; cone or cone-rod dystrophy, X-linked; congenital stationary night blindness, autosomal dominant; congenital stationary night blindness, autosomal recessive; congenital stationary night blindness, X-linked; Leber congenital amaurosis, autosomal dominant; Leber congenital amaurosis, autosomal recessive; macular degeneration, autosomal dominant; macular degeneration, autosomal recessive; ocular retinogenesis disease, autosomal dominant; optic atrophy, autosomal dominant; optic atrophy, autosomal recessive; optic atrophy, X-linked; retinitis pigmentosa, autosomal dominant; retinitis pigmentosa, autosomal recessive; retinitis pigmentosa, X-linked; X-linked; Usher syndrome, autosomal recessive; autosomal dominant retinoschisis; autosomal recessive retinoschisis; mitochondrial retinoschisis; and X-linked retinoschisis, as well as retinoschisis associated with symptomatic / systemic diseases.For example, see RetNet, the Retinal Information Network, available at sph.uth.edu / retnet / home.htm.

[0031] Generally, the method includes administering a therapeutically effective amount of the hPRP or EV described herein, for example, by intravitreal administration, such as by subconjunctival, intracameral or intravitreal injection; see, for example, Amo et al., Prog Retin Eye Res. 2017 Mar;57:134-185; Yamada and Olsen, Dev Ophthalmol. 2016;55:71-83. For example, purified and lyophilized EVs may be stored at -80°C and may be resuspended, for example, in a surgical theater, prior to administration, such as by intravitreal injection, using a 31-gauge needle. EVs can be resuspended in a physiologically acceptable buffer, such as PBS, or in a polymeric scaffold described herein. Dissociated hPRP cells added with stem cells, for example, in a hydrogel (see, for example, a composition containing gelatin-hyaluronic acid gel, such as gelatin hydroxyphenylpropionic acid (gelatin-HPA) and hyaluronic acid-tyramine (HA-Tyr) as described herein (see, for example, WO 2021 / 113515 pamphlet and Dromel et al., NPJ Regen Med. 2021 Dec 20;6(1):85)), or in a retrievable osmotic capsule, can be delivered, for example, through the retina after pars plana vitrectomy (PPV) or through a scleral approach without vitrectomy (Hinkle et al., Stem Cell Research & Therapy volume 12, Article number: 538 (2021); Falkner-Radler et al., The British journal of ophthalmology. 2011;95(3):370-5; Bhattacharya et al., Curr Mol Biol Rep. 2017 Sep; 3(3): 172-182).

[0032] Approximately 3 μg of lyophilized EV was delivered in 3 μL per injection in rodent studies. Clinically used dosing concentrations, amounts, and frequencies can be determined based on animal and clinical trial data.

Examples

[0033] The present invention is further described in the following examples, which do not limit the scope of the present invention as described in the claims.

[0034] Materials and Methods The following materials and methods were used in the following examples.

[0035] Cell culture and isolation process of the rod Human fetal arrival: Fetal eyes arrive within 24 hours in a double box. Incision was started immediately to maintain a high survival rate.

[0036] Incision and dissociation: Incision of the retina was performed on all eyes. The retina was then dissociated with papain in an incubator for 30 minutes. The cell suspension was then centrifuged and seeded into T25 or T75 flasks (depending on the number of cells) coated with fibronectin.

[0037] 1 - 2 weeks of culture: Cells were cultured for 1 - 2 weeks in a 2D layer under hypoxic conditions (5% O2) using FDA - approved medium (animal - product - free; described below). When confluence (about 10 million cells) was reached, the cells were passaged and centrifuged to start sorting.

[0038] Sorting using CD73 / CD276 (PE and APC): Cells were stained with CD73 - PE and CD276 - APC for 30 minutes at 2°C. After washing and preparing the cell sorter, the cells were sorted by gating about 20 - 30% of the positive population. Analyses were performed before and after sorting.

[0039] Culturing to reach high yields: The final cell line was cultured for 4 weeks using the same medium and flasks to obtain approximately 40 million cells for use in experiments or for creating a cell bank.

[0040] Culture medium DMEM / F12 - 500 ml Knockout Serum Replacement (KSR) - 55 ml - 10% 100x l - Glutamax 5.5 ml - 1% 100x Non - Essential Amino Acids - 5.5 ml - 1% Sodium Pyruvate - 5 ml - 1% β - Mercaptoethanol - 5 μl - 0.001% rhEGF (peprotech) - 1000 μl (10 μg / 1 ml) - 0.2% rhFGF (peprotech) - 500 μl (10 μg / ml) - 0.1%

[0041] Eyecup collection 1. The eyeball was placed in the buffer (HBSS / PBS) in a Petri dish, and the retina was carefully dissected without including the RPE or ciliary body by removing the lens and vitreous humor. 2. The retina was suspended in 10 ml of papain solution at 37 °C for 30 minutes to dissociate the tissue into a single - cell suspension. 3. 20 ml of HBSS was put into a tube, and the sample was centrifuged at 2000 rpm for 5 minutes. 4. The buffer was aspirated, the precipitate was resuspended in 1 ml of medium, and then seeded into a T - 75 flask for further expansion culture.

[0042] Cell preparation for sorting 5. The cells were lifted from the confluent flask using trypsin (1:6 in HBSS). 6. The cells were incubated with trypsin at 37 °C for 3 - 4 minutes. 7. The cells were collected into a 50 - ml tube, and approximately 40 ml of medium containing KSR was added to the cell suspension. 8. The suspension was centrifuged at 1200 rpm for 5 minutes at 15 °C. 9. The supernatant was aspirated and the cell pellet was resuspended in 200 μl of Miltenyi running buffer (catalog number 130-107-207).

[0043] Staining for pure rod photoreceptor sorting 10. The cells were incubated on ice for 30 minutes in CD73 PE (catalog number 130-095-183, Miltenyi), CD276 antibody, anti-human, VioBlue® (catalog number 130-099-998). 11. After incubation, the cells were resuspended in 10 ml of HBSS and filtered using a 30-μm filter (Miltenyi). 12. Next, the samples were centrifuged at 300 g for 5 minutes to wash away the antibody. 13. During this time, 1 ml of filtered TYTO buffer in the input chamber was prepared by injecting it into the TYTO cartridge. The cartridge was pressurized to move the buffer to the positive and negative chambers. 14. The buffer was removed from the input chamber to finish cartridge adjustment. 15. Next, the supernatant was discarded and the cells were resuspended in 5 to 10 ml of TYTO buffer according to the cell count (4 to 6 million cells in 5 ml and more than 5 million cells in 10 ml) and injected into the input chamber of the TYTO cartridge for sorting. 16. 100 μl of the cell suspension was placed in an Eppendorf for MACSQUANT analysis before performing TYTO sorting.

[0044] Culture after sorting 1. Using a fine-tip pipette, the cells in the positive chamber were removed and the chamber was flushed with HBSS. 2. 50 μl of the cell suspension was placed in an Eppendorf for MACSQUANT analysis after sorting. 3. The remaining cell suspension was washed by centrifugation at 300 × g for 5 minutes using 15 ml of HBSS. 4. Resuspend the cell pellet in the culture medium and seed it into a fibronectin-coated T-25 or T-75 flask. 5. Incubate at 37 °C under hypoxic conditions (5% O2). 6. Maintain the flask without changing the medium for 48 hours. 7. After 2 days of incubation, change the complete medium. 8. Thereafter, add fresh medium every 2 days until confluence is obtained. 9. Trypsinize the confluent flask (which should become confluent in 7 to 10 days) and seed the cells into 1 T-75 for further expansion culture. 10. Change the medium daily until confluence is reached in 3 to 4 days.

[0045] Cell sorting and analysis Sorting strategy Before running TYTO, set the cells analyzed using MACSQUANT as a control for sorting. Analyze using MACSQUANT by adjusting the voltage so that the selection markers, CD73 and CD276, place the desired population in the quadrant with the highest intensity. After implementing this first gating strategy, place the TYTO cartridge in the device and start the microfluidic flow.

[0046] First, pass the cells through the negative chamber to stabilize the pressure (approximately 150 hPa) and also through the flow of cells in front of the laser (40 ms between two cells). To know the speed in the device, the cells must be stained with at least two different fluorescent dyes. In this way, the device can calculate the time it takes for the cells to go from the first laser to the second one, thereby measuring its speed. As soon as both of these variables are stable, the laser can be started, and the fluorescence data begins to show that approximately 3000 - 5000 cells are passing in front of the laser. Each cell passes in front of three lasers, and its fluorescence intensity is measured and reported in a graph.

[0047] The same gating strategy used as a control for MACSQUANT was also reported for the survival TYTO sorter and sorting was initiated. However, the valve speed and actuation delay had to be set appropriately to capture each cell properly. Backscatter was fit for each cell population (BSB and BSV) flowing through the microfluidic device and the threshold for both BSB and BSV was set at 10 2 ~5.10 2 For PE and VioBlue, the threshold and gating channels were centered around the high fluorescence population, approximately 10 2 to 10 3 The gating time was set at 40 ms at a pressure of 150 hPa. These settings may be changed according to cell size, shape, granularity, intensity, hardness, concentration and can be changed by analyzing cell staining and populations prior to starting sorting. Overall, the goal is to modify the parameters to obtain a high fluorescence population of approximately 10 2 to 10 3 in both the PE and VioBlue lasers that can be sorted.

[0048] During the entire sorting process (usually about 2 hours), the percentage of sorted, gated, positive cells was measured and reported as a function of time. At the end of sorting, the cartridge was removed from the device and the cells were returned to the cell culture for the next step.

[0049] Flow cytometry hPRP (5 x 10 5 cells / mL in medium) was trypsinized and the recovered cell pellet was processed for phenotype and then analyzed using a flow cytometry assay.

[0050] Flow cytometry was performed using a MACSQUANT flow cytometer (Miltenyi, San Diego) (100). Cells were harvested and fixed with paraformaldehyde at 4°C for 15 minutes. The cells were then washed in wash buffer (BD Biosciences) and incubated in block buffer (Pharmingen staining buffer containing 2% goat serum) at room temperature for 30 minutes. The blocked cells were seeded into flat-bottom 96-well plates (treated, sterile, polystyrene, Thomas Scientific), and conjugated primary antibodies (DAPI-Vioblue, CD73-PE, CD276-APC, cone arrestin-FITC, blue opsin-FITC, rhodopsin-FITC, CRX-APC, recoverin-APC, calbindin-FITC, PkCa-FITC, Brn3a-FITC, Ki67-APC, Thy1.2-APC, vimentin-FITC, cynoxin (Cynoxin)-APC, PAX6-APC, NRL-APC, Cmyc-FITC) were used to stain overnight at room temperature. The primary antibodies were diluted in 200 μL of antibody buffer (TBS, 0.3% Triton X-100 and 1% goat serum). After washing the cells three times for 15 minutes each, the secondary antibodies, which were goat-derived anti-rabbit and anti-mouse, were diluted 1:200 in antibody buffer (Jackson Immunoresearch Laboratory). The secondary antibodies were added and left at room temperature for 3 hours. The light scattering and fluorescence signals from each well were measured using a MACSQUANT flow cytometer (2x10 5 events were recorded). The results were analyzed using MACSQUANTify software (Miltenyi). A DAPI-positive single-cell population was gated for each primary antibody. The percentage of positive cells in the gated population was estimated by comparison with blanks and species-specific isotype controls.

[0051] Immunohistochemistry (IHC): Surviving hPRP was grown on chamber slides, and frozen sections from the left eyes of Long Evans were fixed with 4% paraformaldehyde in 0.1 M PBS (Irvine Scientific) for 20 minutes at room temperature. These fixed cells and sections were blocked and permeabilized with blocking solution [(Tris-buffered saline (TBS), 0.3% Triton X-100 and 3% goat serum (Jackson Immunoresearch Laboratories, West Grove, PA)] for 15 minutes. The samples were then rinsed twice with 0.1 M TBS buffer for 15 minutes each, mounted on polylysine microscope slides, and incubated overnight at 4°C with primary antibodies (DAPI-Vioblue, CD73-APC, CD276-Vioblue, Rhodpsin-Cy3, Recoverin-FITC, PKCα-Cy3, CRX-APC, Ki67-Cy3, PAX6-FITC) at concentrations determined in the laboratory. The next day, the samples were rinsed three times with TBS for 15 minutes. Secondary antibodies (goat-derived anti-mouse and anti-rabbit) were added for 1 hour at room temperature. The samples were then washed one last time with TBS before mounting on polylysine microscope slides using lox viscosity slide mounting medium. Digital images were obtained using an Epifluroscent microscope with a 20x objective lens. The electronic image files were managed using Matlab software.

[0052] In vivo evaluation of cells and EV effects In-vivo transplantation Fifteen rd-1 (C3H / HeOuJ) (3-week-old, approximately 15 g in weight) were used as recipients in the experiment. The transplantation was performed in non-immunosuppressed mice. The mice were sedated with intraperitoneal injections of ketamine (100 - 200 mg / kg) and xyalzine (20 mg / kg) for anesthesia. The eyes were first anesthetized using topical proparacaine (0.5%), followed by Genteal to maintain the moisture of the lens during the surgery.

[0053] hPRP or rod - Exo single - cell injections were administered to recipient mice into the subretinal space and intravitreally. Conjunctival incisions and sclerotomies were performed using a precision disposable microsyringe. Cells were injected into the subretinal space using a glass pipette (inner diameter, 150 μm) attached to a 50 - μL Hamilton syringe via a polyethylene tube. hPRP was injected into retinal blebs as a single - cell suspension in PBS. All samples contained approximately 1x10 5 cells and the injection volume was 2 μL for all replicates. Similarly, intravitreal injections were performed using a 31 - g needle with 3 μL of cells in PBS. For subretinal injection, the presence of blebs was examined using a coverslip applied to the eye. A subretinal injection was considered successful when a bleb that glowed under the incision was visible with the operating microscope. Triple antibiotic (Bac / Neo / Poly) was applied topically at the end of the surgery to prevent further infection. The mice were then caged for a 21 - day study.

[0054] The research protocol was reviewed and approved by the Schepens Eye Research Institute Animal Facility and conformed to the Association for Research in Vision Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

[0055] Tissue processing At 8, 15, and 21 days after transplantation, rats were sacrificed by 2 - minute CO 2 asphyxiation. The eyes were enucleated and placed in 4% paraformaldehyde for 24 hours. Subsequently, the tissues were placed in Sorensen's phosphate - buffered solution with increasing concentrations of sucrose (5%, 10%, 20%) to saturation. The eyes were placed in 30% sucrose overnight or until sectioning. The tissues were embedded in cryostat section gelatin medium overnight and sectioned at 15 - μm thickness using a cryostat.

[0056] For subretinal injection, during the sectioning process, every fifth section was stained and examined by epifluorescence for the presence of hPRP using TRA-1-85-FITC and STEM121-FITC (human cell markers), rhodopsin-PE and recoverin-APC (host photoreceptor markers) and DAPI-Vioblue (cell nuclei). Sections were observed under a Lecia Sp8 confocal microscope for transplantation and cell survival in both the subretinal and vitreal cavities of the mouse.

[0057] For intravitreal injection, during the sectioning process, every sixth section was stained with H&E and observed under brightfield microscopy for the morphology and structure of the retina along with the thickness of the photoreceptor layer. To measure the promising neuroprotective effect of hPRP, the outer granular layer of photoreceptors was blindly counted. Frozen vials of the medium were kept at -80°C before use. Prior to performing isolation and lyophilization of EVs, frozen vials of the medium were analyzed in Dr. Redenti's laboratory and full proteomics and miRNA analyses were performed. A common protocol for EV isolation and analysis was carried out.

[0058] Rod-Exo manufacturing process The manufacturing process is summarized in Figure 3. EVs secreted by hPRP cells were obtained from their culture medium. The medium was frozen and then the EVs were isolated by ultracentrifugation to discard debris, dead cells and other unwanted components. The final pellet was resuspended in PBS, lyophilized and stored to keep the EV structure intact. Finally, the EVs were resuspended in water and injected into the vitreous of the eye. hPRP secreted EVs throughout the manufacturing and culturing process. These EVs were released into the culture medium. The inventors stored this medium in approximately 40 - 50 mL tubes and froze it after immunoselection (sorting) and after two passages. Vials of this medium were selected and contained the secreted and selected EVs.

[0059] Isolation of rod-Exo from the medium The frozen medium derived from the fetal hPRP culture (for fetal-derived EVs) was thawed in a water bath (37°C). Ultracentrifugation of the sample followed. First, the sample was spun at 300 g for 10 minutes to discard cells from the raw material. Next was a spin at 2000 g for 10 minutes to discard dead cells, and then a spin at 10,000 g for 30 minutes to discard all cell debris. Finally, double ultracentrifugation was performed to precipitate the EVs (only the supernatant components remained): both were carried out at 150,000 g for 70 minutes. The final supernatant was discarded last to obtain a precipitate of EVs. The EVs were then measured, analyzed, and used for tests and experiments.

[0060] Rod-Exo Lyophilization and Storage The isolated and precipitated EVs were resuspended in PBS (50 mL) to a final volume. Lyophilization by freezing at -80°C was performed to obtain a final powder product. This final powder was stored at -80°C and stored in vials containing 1 g of powdered EV.

[0061] Testing of EVs and Protein Functionality The lyophilized EVs were resuspended in PBS and tested using Western blot, Bradford assay, nanosite, proteomics analysis, and ELISA to examine protein concentration, EV concentration, protein functionality, and the presence of therapeutic proteins. The tests and analysis were compared with pre-lyophilized EVs using Western blot for imaging and analysis of the presence of specific proteins; nanosite was used to analyze EVs and measure concentration; Bradford assay was used to measure total protein concentration; ELISA was used to measure protein functionality; and proteomics analysis was used for the overall analysis of fold changes in proteins.

[0062] In-vivo Tests In this experiment (Example 3), seven 3-week-old mice with a genotype of rho- / - and a body weight of approximately 120 g were used together with four 4-week-old P23 homozygous mice with a body weight of approximately 117 g. No immunosuppressive drugs were given to the mice before transplantation. The mice were injected with ketamine (40 - 80 mg / kg) and xylazine (10 mg / kg) to induce anesthesia. Proparacaine (0.5%) was applied topically to anesthetize the eyes, and gentearl was continued to maintain the moisture of the lens during the operation. The recipient mice were intravitreally injected with HPRP (human progenitor photoreceptor), gel + HPRP, and sham. The described gel was composed of gelatin-hyaluronic acid functionalized with a tyramine side chain and used at a concentration of 5.5% (w / v) (Dromel et al., NPJ Regen Med. 2021 Dec 20;6(1):85). The polymer was cross-linked using 0.1 U / ml of HRP and 1 mM of H 2 O 2 . The cells and gel were injected into the vitreous cavity using a glass pipette (inner diameter, 100 um) attached to a 50-uL Hamilton syringe via a polyethylene tube. All samples contained approximately 75,000 cells, and the gel injection volume was 1 uL for all replicates. Triple antibiotics (Bac / Neo / Poly) were applied topically at the end of the surgery to prevent further infection. The mice were then placed in cages and returned to the normal breeding environment. The research protocol was reviewed and approved by the Schepens Eye Research Institute Animal Facility and followed the Association for Research in Vision Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

[0063] Ophthalmoscope test OptoMotry, designed by Cerebral Mechanics Inc. for rodents, is used to perform optokinetic tracking (OKT) in a non-invasive manner. The inventors placed mice on a platform surrounded by four LCD screens within a light-tight box and used the screens to present visual stimuli to them. To evaluate VA, the mice were presented with vertical sine wave gratings with varying spatial frequencies. The gratings were presented at a fixed distance from the animals, and the minimum spatial frequency that elicited a tracking response was recorded as the VA threshold. VA was measured separately for each eye with an average test time of 20 minutes per animal. For contrast sensitivity, the tracking movement was identified as slow, constant head movements in the direction of the rotating gratings. The spatial frequency threshold was measured by testing the mice at various spatial frequencies between 0.064 and 0.514 cycles / degree with an average test time of 20 minutes per animal. The OptoMotry device uses a proprietary algorithm to correct the test stimulus based on the mouse's tracking reflex. The contrast threshold was measured at a spatial frequency of 0.2 cycles / degree and calculated as the reciprocal of the Michelson contrast. The reciprocal of the contrast threshold was then plotted for analysis.

[0064] Electroretinogram For ERG testing preparation, animals were anesthetized, their pupils were dilated using 1% tropicamide eye drops, and 1 drop of 1% proparacaine followed on the corneal surface. To prevent dehydration, 1 drop of gentearl (corneal lubricant) was applied to the cornea of the untreated eye. In the treated eye, 1 drop of 0.9% sterile saline was applied to the cornea to enable electrical contact with the gold wire loop recording electrode. A 25-gauge platinum needle was inserted subcutaneously in the frontal area to serve as the reference electrode, while another needle was inserted subcutaneously near the tail to serve as the ground electrode. The ERG test was performed by a series of flash luminances created by a Ganzfeld controlled by Diagnosys Espion3. Responses in both dark adaptation (low illumination) and light adaptation (normal illumination) were tested. During the test, short flashes of light were presented to the eyes to stimulate the retina. The electrical responses generated by the retina were recorded by the gold wire loop electrode on the cornea and transmitted to Diagnosys Espion3. The responses were analyzed to determine the function of the animal's retina. This procedure can help diagnose and monitor various retinal diseases and disorders.

[0065] [Example 1] Production of hPRP Fetal retinas were cultured in medium as described above and sorted using a Miltenyi TYTO cell sorter based on the expression of CD73+ and CD276+. The number and morphology of the sorted cells can be seen in Figures 4 and 5.

[0066] 4.2 Sorting of hPRP from Fetal Tissues Figure 6 shows the survival gating strategy and sorting of hPRP from fetal retinas using both PE and VioBlue lasers for CD73- and CD276-positive cells, respectively. Gates were drawn around the exact population to initiate and isolate the sorting of pure hPRP. After sorting, all populations were analyzed (input, positive, and negative fractions). Figure 7 shows the percentage of positive hPRP in each population, reaching more than 95% in the positive chamber of TYTO.

[0067] To confirm the success of the entire sorting process, the trigger rate, sorting rate, and the percentage of cells sorted successfully are monitored and analyzed throughout the process. Figure 8 shows the evolution of these variables during a 1-hour sorting. Finally, the final population of hPRP is analyzed with a MACSQUANT flow cytometer to accurately measure the number of positive cells (CD73+ / CD276+) captured in the positive chamber, compared to the input of the fetal retina (Figure 9).

[0068] 4.3 Characterization of hPRP After sorting and 4 weeks of culture, hPRP was analyzed for their phenotypes using both flow cytometry and immunohistochemical staining (shown in Figures 10 - 13). Figure 10 represents important markers for the high purity of rods in hPRP (CD73, CD276, and recoverin). These markers were found to be higher than 95% in all cell populations after culture and sorting. Analysis of a number of retinal markers was performed using flow cytometry to demonstrate the presence of rods and the absence of all other retinal cells (Figure 11). The presence of high expression of rod markers confirmed the purity of hRPR, while all other retinal cell markers (RGC, bipolar, amacrine, horizontal, cone, and Müller cell markers) were found to be negative, ensuring high rod purity. To confirm these results, hPRP was stained with the same markers and imaged under a fluorescence microscope to demonstrate their expression (Figures 12 - 13). High expression of rod and photoreceptor markers was found, while other retinal cell markers were absent in hPRP.

[0069] 4.4 In vivo hPRP injection into the vitreous and subretinal space To demonstrate the effect of hPRP in retinal neuroprotection, cells were injected into the vitreous and subretinal space of rd1 mice (a well-characterized mouse model of retinal degeneration). Cells were injected at P25 and the animals were sacrificed and analyzed 7 and 21 days after transplantation. To demonstrate neuroprotection, the retina was stained with a cone arrestin marker (red) to measure the amount of host cones (which degenerate at a regulated rate) remaining in the retina. Figure 14 shows that the whole-mount staining of these retinas shows the presence of numerous cones on day 21, suggesting a high neuroprotective effect of hPRP injection. The retina was also incised to measure the outer nuclear layer (ONL) size and confirm the presence of photoreceptors in the degenerate rd1 mice. After measurement (Figure 15), numerous cells in the ONL were found in mice that received hPRP in both the subretinal space and vitreous compared to the control. Finally, to test the safety of hPRP injection, intraocular pressure (IOP) was measured throughout the experiment as shown in Figure 16. A normal increase in IOP was found several days after vitreous injection and returned to the normal range (10 - 20 mmHg) 5 days later and did not increase in subsequent experiments.

[0070] The sorting process (CD73 + / CD276 + ) for the purification of rod precursor photoreceptor cells yielded a viable and 95% pure population of rod photoreceptor cells with an approximately 20% Ki-67 index (indicating consistent proliferation).

[0071] These cells can be used for cell replacement, drug discovery and screening, or other uses where photoreceptors or their precursors may be required.

[0072] [Example 2] Isolation and Characterization of Rod-EVs Proteomic analysis of EVs was performed and the fold changes in proteins were compared to fetal day 14 retina. As seen in the Venn diagram of Figure 17, 31 proteins were found only in our EVs, while 361 were found in our EVSs and in the fetal retina.

[0073] Figure 18 summarizes the entire proteome cargo found and analyzed in the EVs of the inventors. The proteins were classified into six main groups: 1. Retina-specific: Proteins involved in the development and regeneration of the retina (cones, photoreceptors, ganglion cells, bipolar cells) 2. Cell protection: Proteins involved in the neuroprotection of cells 3. CNS development: Proteins involved in the development of nerve cells (neurons, axons, synapses) 4. Immune response: Proteins involved in causing an immune response and immune reaction (cytokines, macrophages) 5. Common cell processes: Proteins found in all cells that are involved in normal cell activities (adhesion, proliferation...) 6. EV-specific: Proteins found on the surface of all EVs

[0074] Table 1 shows the major and most important proteins found in each category, along with a description of the effects of those proteins, the genes associated with the proteins, and their activities.

[0075]

Table 1

[0076] To simplify the analysis of all proteins, subcategories were created for each of the six different established groups. The analysis of the effects and activities of each protein was performed using the protein database (Uniprot) and the gene database (GeneCard). Figure 19 shows the fold changes of all proteins classified into all subcategories for the entire proteome cargo of the inventors' EVs. It is notable that 42 proteins were found to be related to retina-specific development, 80 to cell protection, 51 to CNS development, and only 26 to immune response.

[0077] The inventors performed the first comparison of proteomic cargo between their EVs and 14-week fetal retinal EVs. This comparison characterized specific and beneficial therapeutic agents, but more importantly, immune trigger proteins. As seen in Figure 20, the most common cellular processes and EVs have similar expression in the fetal retina and the inventors' EVs. When comparing the fetal retina with the inventors' EVs, on average, for retinal-specific proteins. However, neuroprotective proteins are extremely upregulated in the inventors' EVs compared to the fetal retina. Most importantly, there is the presence of well-known proteins involved in numerous failures in cell therapy (TGFB1) and the immune response (JCHAIN). In the analysis, the inventors found that these proteins are expressed 10,000-fold less in their EVs compared to the fetal retina. Notably, other immune trigger proteins were found at the same levels in the fetal retina and the inventors' EVs.

[0078] The last analysis performed on the proteomic cargo of the inventors' EVs showed that the proteins are present only in the inventors' EVs and absent in the fetal retina. As seen in Figure 21, while 31 proteins were found to be absent in the fetal retina, they were highly expressed in the inventors' EVs. Notably, there were no immune trigger proteins found only in the inventors' EVs or absent in the fetal retina, suggesting that these immune trigger proteins are common proteins found in most stem cell therapies.

[0079] Table 2 shows the complete list of proteins (402) found in EVs and analyzed as described above.

[0080]

Table 2-1

[0081]

Table 2-2

[0082]

Table 2-3

[0083]

Table 2-4

[0084]

Table 2-5

[0085]

Table 2-6

[0086]

Table 2-7

[0087]

Table 2-8

[0088]

Table 2-9

[0089]

Table 2-10

[0090]

Table 2-11

[0091]

Table 2-12

[0092] [Table 2-13]

[0093] [Example 3] In vivo evaluation of the rod-EV Rho− / − mice are knockout mice lacking the gene for rhodopsin, a light-sensitive protein found in the rod photoreceptors of the retina. Rhodopsin is essential for vision under low light conditions. Thus, Rho− / − mice lose their vision in the dark and their rods do not function properly. They are commonly used in research on retinal degeneration. On the other hand, P23h homozygous mice have a mutation in the rhodopsin gene that results in the production of a defective form of the rhodopsin protein. This mutation is similar to that found in humans with autosomal dominant retinitis pigmentosa (RP). P23h homozygous mice have progressive retinal degeneration and vision loss similar to RP patients. They are used in research to understand the molecular mechanisms underlying RP and to test promising treatments. Therefore, both rho− / − and P23h homozygous mice are used in vision-related research, while their genetic modifications and related characteristics are different.

[0094] Due to this mutation, rod photoreceptor dysfunction begins to degenerate immediately at 3 weeks after birth. This is followed by the death of cones starting at 5 - 6 weeks after birth. To protect the retina from nerve damage, the inventors focused on cone neuroprotection and administered to 4-week-old mice a technical cell + gel (the gel is composed of gelatin and hyaluronic acid combined at a concentration of 5.5% w / v, HRP, and H 2 O 2(crosslinked using) were injected. The inventors tested the function and retinal behavior of these mice weekly after injection. The functional behavior test, Figures 22A - B, was performed using the optomotor test to measure both visual acuity and contrast sensitivity. The inventors confirmed, as shown in a previous paper (Xiao et al., Invest Ophthalmol Vis Sci. 2019 Oct 1;60(13):4196 - 4204), that the visual acuity in Rho - / - does not decline more than 12 weeks prior to the injection group regardless. However, the inventors observed a decline in contrast sensitivity in the SHAM group immediately 2 weeks after injection. The cell + gel treatment was able to stop and delay this decline by at least 1 month with a clear difference seen at 8 and 9 weeks. This is an ongoing study, but the inventors expect to obtain high durability in neuroprotecting the functional behavior of these mice up to at least 12 - 14 weeks.

[0095] These results, as seen in Figures 23A - C, were confirmed by cone function tests performed using electroretinogram (ERG). The inventors focused on the light - adapted B - wave, which is a representative cone function in the retina. As seen in Figure 23A, a significant increase in cone function was observed 7 and 8 weeks after injection, measured using percent increase (Figure 23B), and confirmed using ERG traces (Figure 23C).

[0096] Overall, this ongoing study suggests that the inventors' gel + cells can neuroprotect the function of cones in the retina of degenerated Rho - / - mice.

[0097] As described in the method, P23H has a genetic mutation associated with misfolding of the rhodopsin protein. The inventors recognized that P23H has a more rapid denaturation profile and conducted a similar study on Rho− / −. Although a dark adaptation wave was not observed in P23H, the inventors were able to measure the light adaptation B-wave of three different groups (SHAM, cells, cells + gel) using ERG, as seen in FIGS. 24A–B. A significant improvement in the light adaptation B-wave was observed immediately after 1 week of injection for both cells and cells + gel compared to the SHAM group.

[0098] These results demonstrate the effectiveness of this treatment even for late-stage degenerated retinas.

[0099] Other embodiments The present invention has been described in conjunction with its detailed description, but it is understood that the foregoing description is intended to be illustrative and does not limit the scope of the invention as defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method for providing a population of rod precursor photoreceptor cells (PRPs): A step of providing an early population containing retinal cells derived from mammals, preferably mammals in the embryonic stage; A step of isolating cells expressing CD73 and CD276 from the initial population to provide a substantially purified population of PRP; and A step of culturing the substantially purified population by choice. Methods that include...

2. The method according to claim 1, wherein the step of isolating cells expressing CD73 and CD276 includes optionally using fluorescence-excited cell sorting (FACS) or magnetically activated cell sorting (MACS) in a microfluidic device.

3. The method according to claim 1 or 2, further comprising the step of culturing the initial population, including retinal cells, before isolating cells expressing CD73 and CD276.

4. The method according to claim 1, wherein a substantially purified population of PRP is cultured in a medium containing serum or a serum substitute, EGF and FGF.

5. The method according to claim 1, wherein the substantially purified population of PRP is cultured under hypoxic conditions.

6. The method according to claim 1, wherein the initial population including retinal cells is derived from a mammal in the embryonic stage.

7. The method according to claim 1, wherein the mammal is a human.

8. A substantially purified population of PRP produced by the method of claim 1, preferably comprising at least 75% of cells expressing CD73 and CD276.

9. A substantially purified population of PRP according to claim 8, for use in the treatment of subjects having conditions associated with the loss of retinal rods or cone photoreceptors.

10. A method for treating a subject having a condition associated with loss of retinal rod photoreceptors, comprising the step of administering a therapeutically effective amount of PRP obtained by the method of claim 1 to the subject, preferably comprising cells in which at least 75% of the population expresses CD73 and CD276.

11. A method for providing extracellular vesicles (EVs) from rod photoreceptor progenitor cells, comprising: A step of providing a substantially purified population of PRP according to claim 8; A step of maintaining a substantially purified population of PRP in a culture medium, Preferably, the step of contacting the culture medium with the PRP in the culture for at least 12, 18, 24, 36, 48, or 72 hours; A step of removing the culture medium from the culture; and The process includes isolating EV from the culture medium, method.

12. The method according to claim 11, wherein the step of isolating EV includes the step of ultracentrifugation of the culture medium to obtain a precipitate containing EV.

13. The method according to claim 11 or 12, further comprising the step of freeze-drying the isolated EV.

14. A composition comprising EV obtained by the method of claim 11.

15. The composition according to claim 14, wherein the EV is in a polymeric scaffold.

16. The composition according to claim 15, wherein the polymeric scaffold comprises polycaprolactone (PCL), polylactic acid-glycolic acid (PLGA), polyethylene glycol (PEG), polylactic acid (PLA), polyetherimide (PEI), PNIPAAM, or hydroxyethyl methacrylate.

17. The composition according to claim 14 for use in the treatment of subjects having a condition related to the loss of retinal rods or cone photoreceptors.

18. A method for treating a subject having a condition associated with the loss of retinal rods or cone photoreceptors, comprising the step of administering to the subject a therapeutically effective amount of PRP obtained by the method of claim 11.

19. The present invention according to any one of claims 9, 10, 17, or 18, wherein the condition associated with the loss of retinal rods or cone photoreceptors is, optionally, a hereditary retinal degenerative disease (IRD), cone-rod dystrophy, retinitis pigmentosa (e.g., LCA), or Stargard disease; or a macular degeneration, e.g., atrophic macular degeneration.

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