reviving the cryopreserved day 32 hesc-derived photoreceptor progenitors

焕活视力：干细胞疗法治疗视网膜变性

Inherited retinal diseases and age-related macular degeneration lead to photoreceptor cell loss and eventual blindness. The retinal photoreceptor is the outer segment layer of the retina comprised of specialized cells responsible for phototransduction (i.e., conversion of light to neuronal signals). The rod and cone photoreceptor cells are adjacent to the retinal pigmented layer (RPE)1. Photoreceptor cell replacement therapy to compensate the cell loss has been an emerging and developing therapeutic approach. Embryonic stem cells (ESCs)2,3,4, induced pluripotent stem cells (iPSCs)-derived RPE cells, and retinal progenitor cells (RPCs)4,5,6,7,8 were used to restore the damaged photoreceptor cells. Sub-retinal space, a confined space between the retina and the RPE, is an attractive location to deposit these cells to replace damaged photoreceptor cells, RPE, and Mueller cells due to its vicinity9,10,11.
Gene and cell therapies have been utilizing the sub-retinal space for regenerative medicine for various retinal diseases in pre-clinical studies. This includes the delivery of functional copies of the gene or gene editing tools in the form of either anti-sense oligonucleotide therapy12,13 or CRISPR/Cas9 or base editing via adeno-associated virus (AAV) based strategy14,15,16, implantation of materials (e.g., RPE sheet, retinal prosthetics17,18,19) and differentiated stem cell-derived retinal organoids20,21,22 to treat retinal and RPE-related diseases. Clinical trials using hESC-RPE31 in the sub-retinal space to treat RPE65-associated Leber congenital amaurosis (LCA)23,24, CNGA3-linked achromatopsia25, MERTK-associated retinitis pigmentosa26, choroideremia27,28,29,30 have been proven to be an effective approach. Direct injection of cells to the vicinity of the damaged area greatly improves the chance of cell settlement at the appropriate region, synaptic integration, and eventual visual improvement.
Even though sub-retinal injection in human and large-eyed models (i.e., pig32,33,34,35, rabbit36,37,38,39,40, and non-human primate41,42,43) has been&#160;established, such injection in the murine model is still challenging due to the constrain of the eyeball size and enormous lens occupying the mouse eye44,45,46. However, genetically modified models are only readily available in small animals and not in large animals (i.e., rabbits and non-human primates), therefore sub-retinal injection in mice draws attention to investigate novel therapeutic approaches in retinal genetic disorders. Three major approaches are being used to deliver cells or AAVs into the sub-retinal space, namely the trans-corneal route, trans-scleral route, and the pars plana route (See Figure 2). Trans-corneal and trans-scleral routes are associated with cataract formation, synechiae, choroidal bleeding, and reflux from the injection site11,44,45,47,48,49. We adopted the pars plana approach as a direct visualization of the injection process, and the injection site can be achieved in real-time under the microscope.
We recently described a method that can differentiate human embryonic stem cells (hESCs) into photoreceptor progenitors under xenofree, chemically defined conditions using recombinant human retina-specific laminin isoform LN523. Since LN523 was found to be present in the retina, we hypothesized that the extracellular matrix niche of the human retina could be recapitulated in vitro and thereby support photoreceptor differentiation from the hESCs36. Single-cell transcriptomic analysis showed that photoreceptor progenitors co-expressing cone-rod homeobox and recoverin were generated after 32 days. A retinal degeneration 10 (rd10) mutant mouse model that mimics autosomal human retinitis pigmentosa was used to evaluate the efficacy of the day 32 hESC-derived photoreceptor progenitors in-vivo. The hESC-derived photoreceptor progenitor cells were injected into the sub-retinal space of rd10 mice at P20, where photoceptor dysfunction and degeneration are ongoing36. Here, we describe a detailed protocol for the preparation of the post-cryopreserved hESC-derived photoreceptor progenitors and delivery into the sub-retinal space of rd10 mice. This method can also be used to administer AAVs, cell suspensions, peptides, or chemicals into the sub-retinal space in mice.

作者聚焦：推进视力恢复 - 基于干细胞的视网膜疾病疗法 

rd10小鼠中人胚胎干细胞衍生的光感受器祖细胞的视网膜下递送

My research focuses on the development of stem cell based therapy for advanced stages of retinal diseases that cause photoreceptor degeneration. The goal of our research is to replace the loss of photoreceptors in patients with clinically safe and functional photoreceptor progenitors with the aim of improving their visual acuity or even restoring their vision. The main preclinical model is mice, but their small sized eyeball make it challenging to inject materials, especially under the retina.Developing the skill to perform successful subretinal injections requires patience and perseverance. We have successfully developed a laminin base for the receptor differentiation method that is able to generate photoreceptor progenitors in 32 days. The subretinal transplantations of this photoreceptor progenitors cell suspensions in the rodents resulted in synaptic connectivity between the holes and graft in the eyes.This has led to partial visual restoration. Our approach is simple, the right visualization helps the surgeon handle the needle to the area of interest.

Watch this Scientific Journal Video about Reviving the Cryopreserved Day 32 hESC-Derived Photoreceptor Progenitors at JoVE.com

Reviving the Cryopreserved Day 32 hESC-Derived Photoreceptor Progenitors  

恢复冷冻保存的第 32 天 hESC 衍生的感光祖细胞

首先将PRDM置于37摄氏度的水浴中。从液氮中取出含有第 32 天 hESC 衍生的感光祖细胞的冷冻管，并将其保存在干冰上。然后在 37 摄氏度的水浴中解冻冷冻管三到五分钟。将细胞重悬于一毫升PRDM中。在130G下离心四分钟 除去上清液后，将沉淀重新悬浮在一毫升PRDM中。要计数细胞，取 10 微升细胞悬液，并按照制造商的说明与 0.2% 台盼蓝混合。将细胞混合物移液到细胞计数室载玻片中，并使用自动细胞计数器测定细胞数量和活力。如果细胞活力高于 70%，则将剩余的细胞悬浮液以 130G 离心 4 分钟。除去上清液并将细胞沉淀重新悬浮在PRDM中进行移植。使用 10 μL 移液管吸头，将细胞团块重新悬浮在微量离心管中，直到看不到团块。将悬浮液加载到33号注射器中，并确认细胞溶液通过针头挤出。

reviving-cryopreserved-day-32-hesc-derived-photoreceptor

Research

JoVE Journal

Medicine

Reviving the Cryopreserved Day 32 hESC-Derived Photoreceptor Progenitors

42 次观看.  Singapore Eye Research Institute. 首先将PRDM置于37摄氏度的水浴中。从液氮中取出含有第 32 天 hESC 衍生的感光祖细胞的冷冻管，并将其保存在干冰上。然后在 37 摄氏度的水浴中解冻冷冻管三到五分钟。将细胞重悬于一毫升PRDM中。在130G下离心四分钟 除去上清液后，将沉淀重新悬浮在一毫升PRDM中。要计数细胞，取 10 微升细胞悬液，并按照制造商的说明与 0.2% 台盼蓝混合。将细胞混合物移液到细胞?...

视频: Reviving the Cryopreserved Day 32 hESC-Derived Photoreceptor Progenitors  

Sub-Retinal Delivery of Human Embryonic Stem Cell Derived Photoreceptor Progenitors in rd10 Mice

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal diseases at advanced stages. We have shown human recombinant retina-specific laminin isoform matrix is able to support the differentiation of human embryonic stem cells (hESCs) to photoreceptor progenitors. In addition, sub-retinal injection of these cells has also shown partial restoration in the rd10 rodent and rabbit models. Sub-retinal injection is known to be an established method that has been used to deliver pharmaceutical compounds to the photoreceptor cells and retinal pigmented epithelial (RPE) layer of the eye due to its proximity to the target space. It has also been used to deliver adeno-associated viral vectors into the sub-retinal space to treat retinal diseases. The sub-retinal delivery of pharmaceutical compounds and cells in the murine model is challenging due to the constraint in the size of the murine eyeball. This protocol describes the detailed procedure for the preparation of hESC-derived photoreceptor progenitor cells for injection and the sub-retinal delivery technique of these cells in genetic retinitis pigmentosa mutant, rd10 mice. This approach allows cell therapy to the targeted area, in particular the outer nuclear layer of the retina, where diseases leading to photoreceptor degeneration occur.

We describe a detailed protocol for the preparation of post-cryopreserved hESC-derived photoreceptor progenitor cells and the sub-retinal delivery of these cells in rd10 mice.

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal ...

科研

医学

Begin by placing PRDM in a water bath set at 37 degrees Celsius. Remove a cryovial containing day 32 hESC-derived photoreceptor progenitor cells from liquid nitrogen, and keep it on dry ice. Then thaw the cryovial in a water bath at 37 degrees Celsius for three to five minutes.Re-suspend the cells in one milliliter of PRDM. Centrifuge them at 130G for four minutes After removing the supernatant, re-suspend the pellet in one milliliter of PRDM. To count the cells, take 10 microliters of the cell suspension and mix with 0.2%trypan blue, following the manufacturer's instructions.Pipette the cell mixture into the cell counting chamber slide, and determine the cell number and viability using an automated cell counter. If cell viability is above 70%centrifuge the remaining cell suspension at 130G for four minutes. Remove the supernatant and re-suspend the cell pellet in PRDM for transplantation.Using a 10 microliter pipette tip, re-suspend cell clumps in the microfuge tube until no clumps are visible. Load the suspension into a 33-gauge injection syringe and confirm the extrusion of cell solution through the needle.

Sub-Retinal Delivery of the hESC-Derived Photoreceptor Progenitor Cells in rd10 Mice

Sub-Retinal Delivery of the hESC-Derived Photoreceptor Progenitor Cells in  rd10  Mice  

To begin, take an anesthetized mouse and apply one drop of 1%tropicamide and 2.5%phenylephrine into the eye to dilate the pupil. Then apply an ophthalmic gel to the cornea to prevent dryness and anesthesia induced cataracts. Next, set up an upright operating microscope with a direct light path in a sterile environment to perform the subretinal injection.To prepare the 10 microliter glass syringe, detach the needle hub and attach the 33 gauge blunt needle. Take the metal hub cover and securely fasten the needle onto the syringe. Flush the needle with distilled water to test leakage and needle patency.Then empty the syringe and set it aside carefully. Next, position the anesthetized mouse on a cushion and orient the treatment eye toward the microscope's objective. Apply 0.5%proparacaine hydrochloride and wait for 30 seconds.Then apply 150 microliters of ophthalmic gel to the eye and affix a round cover slip. Perform a preliminary eye examination observing the cornea, iris, pupil, lens, and conjunctiva. Adjust the focal plane to visualize the fundus of the mouse eye through the pupil.Then align the head so that the optic head aligns with the center of the pupil. Afterwards, tap the base of the tube containing HESC derived photoreceptor progenitors to get a uniform cell suspension. Using a 10 microliter glass microliter syringe with a 33 gauge blunt needle, draw two microliters of cell mixture.Use a 30 gauge disposable needle to create a sclerectomy wound positioned two millimeters behind the limbus. Maintain the needle's angle at approximately 45 degrees to prevent contact with the lens. Once the needle tip becomes visible within the eye, carefully retract the needle.Discard the used needle into a sharp bin to prevent needle prick injuries. Now take the glass syringe and insert the blunt needle into the sclerectomy wound. Advance the blunt needle without touching the lens until it reaches the opposite retina of the entry wound.Gently penetrate the retina until a pressure branch on the sclera is seen. Then inject two microliters of the cell suspension or PRDM media into the subretinal space while maintaining gentle pressure on the syringe. The formation of a visible bleb indicates successful injection.Wait 10 seconds to allow the cells to settle down. Gently retract the needle from the eye. To visualize the bleb, move the mouse head to position the eye under the microscope and secure the position by gently holding the head.For intraoperative OCT, use the IOCT function on the operating microscope and select cube on the OCT screen. Position the scanning area on the bleb by pressing the arrow buttons. To adjust the OCT, slide centering and focus for the best OCT quality, press capture or scan to acquire the OCT scan of the bleb area and review the images to check the quality of the scans.Finally, remove the cover slip and gently clean the gel from the eye using gauze. Administer antibiotic ointment once after the injection to prevent infection. The OCT scan revealed distinct HESC entities suspended in the cell treated eye.In contrast, the media treated eye displayed unclouded fluid, void of cells within the subretinal space.