RU2730937C1 - Method of transplantation of retinal pigment epithelium (rpe) cells differentiated from induced human pluripotent stem cells, with retinal pigment epithelium atrophy - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, namely to ophthalmology, and can be used for transplantation of cells of retinal pigment epithelium differentiated from induced pluripotent stem, at retinal pigment epithelium (RPE) atrophy in experiment on rabbits. Induced retinal detachment according to the area of RP atrophy and through a linear section of retina in the area of detachment is introduced subretinal induced pluripotent stem cells (IPSC) RPE, fixed in form of monolayer on substrate with thickness of 50 mcm, obtained by electrospinning and consisting of 50 % of nanofibres of hydrophilic polyurethane and 50 % of gelatin.EFFECT: method enables improving functional activity of the retina in eyes with RP atrophy, creating bases in the form of a substrate for providing adequate adhesion of RPE stem cells in a polarized monolayer, which will increase engraftment and functioning of IPSC-RPE.1 cl, 1 ex, 4 dwg

Description

The invention relates to medicine, namely to ophthalmology, and can be used for subretinal transplantation of retinal pigment epithelium (RPE) cells, differentiated from induced human pluripotent stem cells (hereinafter - iPSC-RPE), for the treatment of retinal diseases accompanied by atrophy of the retinal pigment epithelium ...

Many forms of vision-threatening diseases, including age-related macular degeneration, are caused by dysfunction, degeneration, and loss of RPE. The treatment options for degenerative diseases of the retina are extremely limited, therefore, at the present stage, new approaches are being actively developed on experimental models for the treatment of this group of ophthalmic diseases. Currently, transplantation of iPSC-RPE is one of the most promising areas, both in terms of studying the molecular mechanisms of cellular pathologies in a personalized approach, and developing approaches to cell therapy for various degenerative retinal diseases [Rao, M., Gottesfeld, J.M. (2014) Introduction to thematic minireview series: Development of human therapeutics based on induced pluripotent stem cell (iPSC) technology, The Journal of Biological Chemistry, 289, 4553-4554 .; A.E. Kharitonov, A.B. Surdina, O.S. Lebedeva, A.N. Bogomazova, M.A. Lagarkov. Possibilities of using pluripotent stem cells to restore damaged retinal pigment epithelium. ACTA NATURAE | VOLUME 10 No. 3 (38) 2018].

There is a method in which differentiated iPSC-RPE are transplanted subretinally in the form of a cell suspension equivalent to 50 thousand in the eyes of rabbits with induced RPE atrophy. Preliminary results have confirmed the safety of this approach, however, data on efficacy, including functional integration of transplanted cells, have not yet been demonstrated [Petrus-Reurer S. et al. Integration of subretinal suspension transplants of human embryonic stem cell-derived retinal pigment epithelial cells in a large-eyed model of geographic atrophy // Investigative ophthalmology & visual science. - 2017. - T. 58. - No. 2. - C. 1314-1322.]

Transplantation of iPSC-RPE in suspension form is widely used due to the lighter technique, but it is at a disadvantage due to poor cell survival due to apoptosis, since these cells do not find proper mechanical support. Transplantation of RPE cells on a substrate provides anatomical integration and reliable cell survival. Until now, there is an active comparison between the two existing methods of RPE cell transplantation in terms of cell survival, anatomical and functional integration [Uebersax E.D., Grindstaff R.D., Defoe D.M. Survival of the retinal pigment epithelium in vitro: comparison of freshly isolated and subcultured cells // Exp. Eye Res. 2000.70. (3). 381-390].

Nymark S, Sorkio A, Wang J-H, et al. (2015) Ultrathin Polyimide Membrane as Cell Carrier for Subretinal Transplantation of Human Embryonic Stem Cell Derived Retinal Pigment Epithelium. PLoS ONE 10(11): e0143669. doi:10.1371/joumal.pone.0143669]The known method of transplantation of RPE cells, differentiated from human embryonic stem cells (ESC-RPE), in which an ultrathin and porous polyimide membrane (PI) is used as a framework, which, together with the cells, was transplanted into the subretinal space of rabbits. The rabbits were under dynamic observation for three months after surgery. According to the data of functional and morphological research methods, despite the correct localization of PI, over time there was a loss and rejection of transplanted cells [Ilmarinen T, Hiidenmaa H,

Nymark S, Sorkio A, Wang JH, et al. (2015) Ultrathin Polyimide Membrane as Cell Carrier for Subretinal Transplantation of Human Embryonic Stem Cell Derived Retinal Pigment Epithelium. PLoS ONE 10 (11): e0143669. doi: 10.1371 / joumal.pone.0143669]

The closest analogue of the present invention is a method of transplantation of ESC-RPE, in which a non-absorbable substrate from parylene-C (CPCB-RPE1) with a monolayer of ESC-RPE was implanted by injection through a sclerotomy and retinotomy incision into the subretinal space of Yucatan minipigs. The animals were euthanized immediately after implantation to study the position of the graft. Although ultra-thin substrates are theoretically good scaffolds for subretinal transplantation due to their permeability, they most often require complex surgical maneuvers, subsequently leading to intraoperative and postoperative complications [https://link.springer.com/article/10.1186/s40942-017-0095 -6].

The choice of substrate is an important parameter for the cultivation and successful transplantation of RPE cells. Currently, the search is underway for an optimal substrate that mimics the structural and functional properties of the Bruch membrane, which provides the basis for RPE cell adhesion in a polarized monolayer, which is cost-effective and safe to use.

The objective of the invention is to select an optimal substrate for transplantation of iPSC-RPE and to develop a technique for their transplantation into the subretinal space of rabbits with a RPE atrophy model.

The technical result of the proposed invention is to increase the functional activity of the retina in eyes with RPE atrophy, to create a base in the form of a substrate to ensure adequate adhesion of RPE cells differentiated from human pluripotent stem cells in a polarized monolayer, which ultimately increases the integration capacity and functioning of iPSC-RPE.

The technical result is achieved due to the transplantation of iPSC-RPEs fixed in the form of a monolayer on a substrate 50 μm thick, obtained by the electrospinning method and consisting of 50% hydrophilic polyurethane nanofibers and 50% of gelatin, into the retinal detachment induced, respectively, by the RPE atrophy zone through its linear incision ...

The method is carried out as follows.

In rabbits with atrophy of the retinal pigment epithelium (RPE), retinal detachment is induced corresponding to the RPE atrophy zone. Through a linear incision of the retina in the detachment zone, subretinal iPSC-RPE is injected, fixed in the form of a monolayer on a 50-μm-thick electrospinning substrate and consisting of 50% hydrophilic polyurethane nanofibers and 50% gelatin.

The support for fixation in the form of a monolayer was obtained by electrospinning. The substrate is 50% hydrophilic polyurethane nanofibers and 50% gelatin. Studies have shown that, when cultivated on this substrate, cells are capable of forming a monolayer, have a good growth rate, hexagonal morphology, and rapid pigment accumulation. Before plating on a substrate, the cells were cultured in DMEM / F12 medium (PanEco) supplemented with 10% fetal bovine serum (Gibco), 2% nonessential amino acids (PanEco), 100-fold GlutaMAX (Gibco) and penicillin / streptopicin (50 U / ml / 50 μg / ml) (PanEco) until a monolayer is reached. Then the cells were seeded with the substrate at a rate of 1: 1 in area (the area of the vial for cultivation to the area of the substrate, approximately 100,000 cells / cm ² ). For inoculation onto a substrate, cells were washed with Hanks solution (PanEco), incubated for 5-7 minutes in 0.05% trypsin in EDTA (Gibco), and centrifuged. Before transplantation, the substrate was transferred into an Eppendorf tube, washed from the medium with phosphate-saline solution (PanEco). (ISO 7198: 1998, "Cardiovascular implants - Tubular vascular prostheses. 1998).

One month before the transplantation of iPSC-RPE, a model of RPE atrophy was created as follows: bevacizumab in a volume of 10 μL containing 0.025 mg of the drug was injected into the subretinal space of rabbits at a distance of 0.5 pd below the optic nerve head with the formation of a subretinal bladder.

Typical morphological signs observed in experimental modeling of retinal atrophy are dystrophic changes in all its layers expressed to varying degrees with impaired histoarchitectonics and stratification, foci of discharge of nuclear layers and layers of nerve fibers, subatrophy and vacuolization of the ganglion cell layer. Changes in the pigment epithelium are represented by foci of balloon dystrophy and destruction with extracellular prolapse and deposition of pigment clumps, destruction and lysis of pigment cells with subtotal atrophy and disappearance of the pigment cell layer. According to the zone of atrophy, on the surface of the choriocapillary layer, a substrate made of polymer material is found, on which the cells of the pigment epithelium are visualized.

For transplantation, animals were anesthetized with intramuscular administration of Zoletil and Xylazine. Midriasis was achieved by instilling Appamide. The operation was performed on the right eye, leaving the paired eye for control. Before the operation, Vigamox and Alkain were topically instilled. Retinal detachment was induced according to the RPE atrophy zone. Through a linear retinal incision in the detachment zone, iPSC-RPE was injected subretinally, fixed in the form of a monolayer on a 50-μm-thick electrospinning substrate and consisting of 50% hydrophilic polyurethane nanofibers and 50% gelatin.

After surgery, all rabbits were injected subconjunctivally with a solution of Gentamicin and Dexamethasone at a dose of 3 mg / kg and 0.2 mg / kg, respectively. In the next 14 days, a solution of Dexamethasone 0.1% was instilled into the conjunctival cavity 3 r / d. The day before surgery and for the next 4 months, the rabbits received Sandimmun neoral (oral solution) 100 mg / ml (calculated as 10 mg per kg of body weight).

Experimental studies were carried out on 10 rabbits (10 eyes) of the albino breed, weighing 2.0 - 2.5 kg. The experiment lasted 4 months. After 4 months, the animals were removed from the experiment by the method of air embolism after the introduction of the rabbit into anesthesia (according to the order of the USSR Ministry of Higher Education No. 724 of 13.11.184). The whole eyeballs were enucleated, after which they were fixed in a neutral 10% formalin solution for 1 day. After fixation, the eyeballs were cut into 3 blocks so that the zone of induced atrophy and the substrate were in the central block. After standard histological tracing, the central pads were embedded in paraffin. Serial sections were prepared from paraffin blocks, which were stained with hematoxylin and eosin according to a standard technique. Survey microscopic examination was carried out on a microscope equipped with a digital camera at a magnification of x400.

Optical coherence tomography was performed in both eyes using Heidelberg Spectralis ™ SD-OCT (Heidelberg Engineering, Germany) before surgery in order to visualize the area of pre-created atrophy. Further, the determined observation period is 2 days, 2 weeks, 1 month and 4 months after surgery.

2 days after surgery, an OCT image showed a zone of atrophy of the retinal pigment epithelium, leading to an increased penetration of the laser beam into the underlying tissues, a hyperreflective zone corresponding to a substrate with IPSC-RPE with an adjacent retina and a rupture, respectively, at the site of retinotomy and localization of the substrate. When carrying out fundus autofluorescence, the zone of hyperautofluorescence corresponding to the localization of the substrate was determined.

After 2 weeks, 1 month and 4 months after surgery, a substrate was determined that was tightly adjacent to the choroid and structures of the neurosensory retina, spontaneous closure of the gap was noted. The study of fundus autofluorescence showed the presence of a hyperfluorescence zone corresponding to the location of the substrate with iPSC-RPE, surrounded by a rim of speckled hypofluorescence corresponding to RPE atrophy.

Example. Rabbit number 5. A model of RPE atrophy was created; after 1 month, iPSC-RPE transplantation on a substrate was performed. The rabbit was euthanized 4 months after implantation and the enucleated eyes were sent for morphological examination. On the histological section, a substrate made of polymer material is visualized, implanted on the Bruch's membrane of a rabbit (indicated by an arrow). Implanted RPE stem cells (circled) are identified on the surface of the polymer substrate, the sclera is indicated by an asterisk - Fig. 1. Before surgery, optical coherence tomography was performed in both eyes in order to visualize the area of pre-created atrophy - Fig. 2. Further, the determined terms of observation - 2 days, 2 weeks, 1 month and 4 months after surgery. 2 days after surgery, an OCT image showed a zone of atrophy of the retinal pigment epithelium and a hyperreflective zone corresponding to a substrate with iPSC-RPE with an adjacent retina and a gap, respectively, at the retinotomy site and substrate localization. When carrying out fundus autofluorescence, a zone of hyperautofluorescence was determined corresponding to the localization of the substrate with iPSC-RPE surrounded by a rim of speckled hypofluorescence corresponding to RPE atrophy - Fig. 3. In 2 weeks, 1 month and 4 months after surgery, a substrate was determined that was tightly adjacent to the choroid and structures of the neurosensory retina, spontaneous closure of the gap was noted - Fig. 4. Studies have shown that transplantation of iPSC-RPE, fixed in the form of a monolayer on a 50-μm-thick substrate obtained by electrospinning and consisting of 50% hydrophilic polyurethane nanofibers and 50% of gelatin, shows a positive effect of transplantation of the substrate with iPSC-RPE on the functional activity of the retina in eyes with atrophy. The implantable substrate is correctly integrated and provides the basis for adhesion of RPE stem cells in a polarized monolayer, which subsequently affects their further engraftment and functioning. Thus, this method makes it possible to improve the technology of iPSC-RPE transplantation at the preclinical stages of the study, which opens up new perspectives in the treatment of degenerative retinal diseases and the possibility of a personalized approach.

Claims (1)

The method of transplantation of retinal pigment epithelium cells, differentiated from induced pluripotent stem cells, with atrophy of the retinal pigment epithelium (RPE) in an experiment on rabbits, characterized in that they induce retinal detachment, respectively, in the RPE atrophy zone and through a linear retinal incision in the zone of retinal detachment, the pluripotent stem cells are injected RPE cells (iPSCs) fixed in the form of a monolayer on a 50-μm-thick electrospinning substrate consisting of 50% hydrophilic polyurethane nanofibers and 50% gelatin.

Images