A method for constructing pacemaker cells and preparing retinal organoids based on ChR2 and GCaMP5G double transfection of hESCs

Abstract

This invention relates to the field of organoid preparation technology, and discloses a method for preparing retinal organoids by constructing pacemaker cells based on ChR2 and GCaMP5G double transfection of hESCs. The method includes the following steps: Step 1, Transfection: Transfecting hESCs with ChR2 and GCaMP5G; Step 2, Screening: Screening for ChR2 and GCaMP5G double-positive cells using FASC; Step 3, Differentiation Culture: Differentiating and culturing the two-dimensional ChR2 and GCaMP5G double-positive hESCs into three-dimensional retinal organoids; Step 4, Photostimulation: Activating ChR2 with 470nm blue light and observing calcium signals under a two-photon microscope. This method, by transfecting hESCs with ChR2 and GCaMP5G, screening for ChR2 and GCaMP5G double-positive cells, and culturing them into retinal organoids, effectively accelerates the differentiation and culture of hESCs into three-dimensional retinal organoids by specific blue light stimulation, thereby effectively improving the speed of retinal organoid preparation and shortening the cycle.

Description

Technical Field

[0001] This invention relates to the field of organoid preparation technology, specifically to a method for preparing retinal organoids by constructing pacemaker cells based on ChR2 and GCaMP5G double transfection of hESC. Background Technology

[0002] Retinal organoids are three-dimensional structures generated in vitro using stem cell technology (such as induced pluripotent stem cells, iPSCs) that mimic the development and function of the human retina. They have important applications in fields such as retinal development research, disease modeling, and drug screening.

[0003] Currently, retinal organoids are typically prepared through the following steps: Step 1, stem cell culture: Embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) are used as starting materials. Step 2, directed differentiation: Stem cells are directed to differentiate into retinal progenitor cells by adding specific growth factors and small molecule compounds. Step 3, three-dimensional culture: Retinal progenitor cells are transferred to a three-dimensional culture system to promote their self-organization into retinal organoids. Step 4, maturation culture: After several weeks to months of culture, the organoids gradually develop a layered structure similar to the retina, including photoreceptor cells (rod cells and cone cells), bipolar cells, ganglion cells, etc. However, the existing technology still has the following drawbacks:

[0004] (1) The preparation of existing retinal organoids requires multiple steps of differentiation, three-dimensional culture and long-term maturation, and the whole process usually takes several months (e.g. Gonzalez-Cordero A, Kruczek K, Naeem A, Fernando M, et al. (2017) Journal article: Recapitulation of Human Retinal Development from Human Pluripotent Stem Cells Generates Transplantable Populations of ConePhotoreceptors). Stem Cell Reports, 9: 1-18. Available from DOI: 10.1016 / j.stemcr.2017.07.022; records require 17 weeks), the steps are cumbersome and the technical requirements are high; and the time consumption and cost are high, which limits its application in high-throughput screening or large-scale research. (2) The existing retinal organoids are not mature enough. Although retinal organoids can simulate the layered structure of the retina, their maturity is still different from that of the real retina, especially the functions of photoreceptor cells and ganglion cells are not fully mature; and the imperfect function of retinal organoids may affect the accuracy of disease modeling, especially for the study of late retinal diseases. (3) The retinal organoids prepared by different batches of the existing methods may have significant differences in cell composition, structure and function, which significantly reduces the reproducibility of the experiment and increases the difficulty of data interpretation. (4) Although the retinal organoids prepared by the existing methods can generate photoreceptor cells, the photosensitivity and electrophysiological function of these cells are usually weak, making it difficult to completely simulate the light signal conversion process of the real retina, thus affecting the results of optogenetic experiments or drug screening.

[0005] Therefore, there is a need to develop a method for preparing retinal organoids that has a high success rate, short cycle, consistent cell maturity, and good sensitivity. This method would not only effectively compensate for the shortcomings of existing technologies but also significantly improve the efficiency and effectiveness of retinal organoid preparation, which is of great significance for advancing related experimental research based on retinal organoids. Summary of the Invention

[0006] The present invention aims to provide a method for constructing pacemaker cells and preparing retinal organoids based on ChR2 and GCaMP5G double transfection hESC, so as to solve the technical problem of long cycle of existing methods for preparing retinal organoids.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: a method for constructing pacemaker cells and preparing retinal organoids based on ChR2 and GCaMP5G double transfection of hESCs, comprising the following steps:

[0008] Step 1, Transfection: Transfect ChR2 and GCaMP5G in hESC;

[0009] Step 2, Screening: ChR2 and GCaMP5G double-positive cells were screened using FASC;

[0010] Step 3: Differentiation and Culture: Two-dimensional ChR2 and GCaMP5G double-positive hESCs were differentiated and cultured into three-dimensional retinal organoids;

[0011] Step 4, photostimulation: ChR2 is activated with 470nm blue light, and calcium signals are observed under a two-photon microscope.

[0012] The principle of this scheme is:

[0013] ChR2 (channelrhodopsin-2) is a light-controlled cation channel. Upon activation, the channel opens, allowing cation influx that depolarizes the cell membrane potential. This depolarization is then conducted to other cells via gap junctions, ultimately forming a calcium wave similar to that observed in the developing retina. GCaMP5G is a gene-edited calcium fluorescent indicator, composed of green fluorescent protein (GFP), calmodulin (CaM), and a peptide segment M13 of myosin light chain kinase. When ChR2 is activated, calcium ions influx and bind to CaM, inducing a conformational change in CaM, causing its light chain region to bind to M13. This enhances the protonation of the GFP chromophore under specific wavelength excitation, leading to increased absorbance and fluorescence intensity, thus enabling the visualization of ChR2-mediated calcium waves. This protocol transfects ChR2 into hESCs. Successfully transfected ChR2-positive cells possess pacemaker cell characteristics and play a crucial role in the entire calcium wave formation process. By activating open channels, it initially excites other cells and effectively mimics calcium waves in the retina, thereby promoting the migration and differentiation of progenitor cells, making the retinal layering more realistic, and allowing more photoreceptors to appear in the normal outer layer of the neuroepithelium, survive and complete functional development, forming more active retinal neural circuits with more neurons, thus further promoting the maturation of the entire organoid.

[0014] The advantages of this solution are:

[0015] 1. Compared with the long cycle of existing technologies for preparing retinal organoids, this method effectively accelerates the differentiation and culture of hESCs into three-dimensional retinal organoids by transfecting hESCs with ChR2 and GCaMP5G, screening for double-positive cells of ChR2 and GCaMP5G, and then culturing them. This effectively improves the speed of retinal organoid preparation and shortens the cycle.

[0016] 2. Compared with the large batch-to-batch variability of retinal organoids prepared by existing technologies, this method can mass-produce retinal organoids through transfection, screening, differentiation culture and photostimulation. Moreover, the resulting retinal organoids have high structural and functional similarity and small batch-to-batch variability, thereby improving the reproducibility of experiments based on retinal organoids, reducing the difficulty of data interpretation, and promoting the rapid development of related disciplines.

[0017] 3. This scheme uses the optogenetic tool ChR2 to construct pacemaker cells and induces their excitation through light control, thereby generating calcium waves, which has a significant impact on promoting the development of retinal organoids and has ideal prospects for widespread application.

[0018] 4. This protocol utilizes the simultaneous transfection of ChR2 and GCaMP5G into hESCs (human embryonic stem cells) to facilitate rapid detection of differentiation culture and activation effects by inducing changes in cell membrane potential and triggering calcium ion influx after ChR2 is activated by blue light. Specifically, GCaMP5G can react with intracellular calcium ions (Ca... 2+ When calcium ion concentration increases, GCaMP5G emits a fluorescent signal. Therefore, in this study, GCaMP5G was transfected into hESCs. In differentiated retinal organoids, the dynamic changes in calcium signals in retinal organoids could be observed in real time using two-photon microscopy, thereby studying intercellular signal transduction and functional activities.

[0019] 5. This protocol simultaneously transfects hESC cells with ChR2 and GCaMP5G, followed by FASC (Fluorescence-Activated Cell Sorting) for selection, facilitating rapid screening for double-positive cells and improving efficiency. Specifically, after successful ChR2 transfection, hESC cells express a red fluorescent marker, and after successful GCaMP5G transfection, hESC cells express a green fluorescent marker. The presence of both red and green fluorescent markers allows FASC to quickly detect, analyze, and sort cells based on their fluorescent labels, thereby achieving rapid screening of double-positive cells and further improving the efficiency of retinal organoid preparation.

[0020] Preferably, as an improvement, in step one, transfection includes the following steps:

[0021] A1. Separate hESCs into single-cell hESCs;

[0022] A2. The hESC in A1 was suspended in E8 medium containing lentivirus LV-CAG-hChR2(H134R)-mCherry-WPRE, Y-27632 and polyethylene to prepare a transfection suspension.

[0023] A3. Plate the transfection suspension onto the surface of the vitronectin coating and incubate for 16-18 hours;

[0024] A4. Discard the viral supernatant, add fresh E8 medium containing the inhibitor Y-27632, and incubate for 20-24 hours before removing.

[0025] A5. Repeat steps A2 to A4, replacing LV-CAG-hChR2(H134R)-mCherry-WPRE in A2 with pLOV-CMV-GCaMP5G to obtain a mixed cell solution containing double transfection of ChR2 and GCaMP5G.

[0026] Technical Effects: This protocol employs the above-mentioned settings to improve the success rate of double transfection. Specifically, transfection with lentiviruses (such as lentivirus LV-CAG-hChR2(H134R)-mCherry-WPRE and lentivirus pLOV-CMV-GCaMP5G) integrates the transfected content into the genomic DNA of the target cells, facilitating its long-term expression. Y-27632 is a ROCK inhibitor that reduces apoptosis and mechanical damage, making it particularly suitable for single-cell suspension culture or post-transfected cells; inhibiting the ROCK signaling pathway also reduces cell adhesion, preventing cell aggregation and thus improving transfection efficiency. Polyethylene is a cationic polymer that can form a complex with DNA, promoting DNA entry into cells. Vitronectin is an extracellular matrix protein containing the RGD (arginine-glycine-aspartic acid) sequence, which can bind to integrin receptors on the cell surface, promoting cell adhesion. In transfection experiments, the vitronectin coating can improve cell adhesion and survival rate, thereby optimizing transfection efficiency.

[0027] Preferably, as an improvement, the Y-27632 content in the E8 culture medium is 10~15μM, and the amount of polyethylene used is 10~15μg / ml.

[0028] Technical Effects: This solution, employing the aforementioned settings, facilitates optimization and improvement of the survival rate and transfection efficiency of difficult-to-transfect cells (such as stem cells). Specifically, through long-term experiments, the applicant discovered that excessively high Y-27632 content can lead to excessive cell dispersion, affecting intercellular signal transduction and function. Furthermore, excessively high Y-27632 levels may interfere with normal cellular physiological functions (such as differentiation capacity), thus impacting transfection efficiency. Conversely, excessively low Y-27632 content may cause cell aggregation due to insufficient inhibition of the ROCK signaling pathway, reducing transfection efficiency; and cells may die in large numbers during transfection due to mechanical damage or stress. Excessive polyethylene dosage may be toxic to cells, leading to cell death; and excessive polyethylene may result in overly large DNA complexes, making it difficult for them to enter cells and reducing transfection efficiency. Additionally, excessive polyethylene may non-specifically bind to the cell membrane, affecting cell function. Conversely, insufficient polyethylene dosage may reduce transfection efficiency due to incomplete DNA complex formation, and may even fail to effectively protect DNA from nuclease degradation.

[0029] Preferably, as an improvement, the transfection fold of both LV-CAG-hChR2(H134R)-mCherry-WPRE and pLOV-CMV-GCaMP5G is 5 to 10 times.

[0030] Technical Effects: This scheme, employing the above settings, facilitates a balance between transfection efficiency and post-transfection cell viability. Through long-term experiments, the applicant discovered that excessively low MOI (transfection fold) leads to low transfection rates, making cell line establishment difficult or resulting in weak photogene function in infected cells; while excessively high MOI leads to loss of cell pluripotency, hindering cell proliferation and survival, or causing organoid differentiation failure.

[0031] Preferably, as an improvement, in step two, the screening includes the following:

[0032] B1. Use papain to dissociate the above mixed cell fluid into single cells;

[0033] B2. After filtering out the single cells from B1, resuspend them in PBS buffer to form a single-cell suspension.

[0034] B3. The above single-cell suspension was sorted using fluorescently activated cell sorting to obtain ChR2 and GCaMP5G double-positive cell suspensions.

[0035] Technical Effects: This protocol, employing the above-described setup, facilitates rapid screening to obtain ChR2 and GCaMP5G double-positive cell suspensions. Specifically, the protocol first uses papain to dissociate cells, obtaining a single-cell suspension while maintaining cell viability. Then, filtration and resuspending in PBS (phosphate-buffered saline) remove clumps and debris, preparing a high-quality single-cell suspension. Finally, FACS sorting yields a high-purity ChR2 and GCaMP5G double-positive cell suspension for subsequent experiments.

[0036] Preferably, as an improvement, in step three, the differentiation culture includes the following steps:

[0037] C1. The double-positive cell suspension obtained in B3 was dissociated into a single-cell suspension. After centrifugation, the cell pellet was resuspended in retinal differentiation medium containing DNase I and Y-27632 to obtain a cell suspension.

[0038] C2. On day 0, add the cell suspension from C1 to each well of a V-bottom 96-well plate with low cell adhesion for incubation.

[0039] C3. On day 6, the culture medium was replaced with fresh retinal differentiation medium containing 1.5 nM BMP4 and cultured for another time. Half of the culture medium was replaced every three days to obtain the embryoid body hERO.

[0040] C4. On day 18, the hERO obtained from C3 culture was transferred to a culture dish and cultured in a long-term culture medium until the neural retinal structure appeared, thus obtaining retinal organoids (ROs).

[0041] C5. The ROs obtained in C4 were further induced to differentiate in a long-term induction medium for 70 days.

[0042] Technical effect: The above settings facilitate the rapid differentiation and culture of double-transfected cells into mature retinal organoids.

[0043] Preferably, as an improvement, the retinal differentiation medium consists of 45 parts IMDM medium, 45 parts F12-Glutamax medium, 1 part chemically defined lipid concentrate, 10 parts serum substitute, 450 μM monothioglycerol, 100 U / ml penicillin, and 100 mg / ml streptomycin; the retinal differentiation medium contains 0.05 mg / ml DNase_I and 20 μM Y-27632; the long-term medium consists of DMEM / F12-Glutamax medium supplemented with 1% N2 supplement, 10% fetal bovine serum, 0.5 μM RA, 0.1 mM taurine, and 0.25 μg / ml Fungizone.

[0044] Technical Effects: This scheme employs the above-mentioned setup. The retinal differentiation culture medium is used to induce pluripotent stem cells to differentiate into retinal cells and form retinal organoids; the long-term culture medium is used to maintain the long-term culture of retinal organoids, promoting their maturation and functional maintenance. Through long-term experiments, the applicant discovered that if the Y-27632 content in the retinal differentiation culture medium is too low, the directed differentiation of retinal progenitor cells will be hindered, leading to a reduction in photoreceptor generation or structural damage; if the dosage of penicillin and streptomycin is too low, insufficient antibacterial ability will result in cell death and abnormal differentiation.

[0045] Preferably, as an improvement, in step four, the light stimulation includes the following: after 70 days of differentiation culture, the cultured ROs are continuously stimulated with 470nm blue light for 50 hours at a frequency of once every 25 seconds, and each stimulation includes a frequency of 100Hz, a duration of 3ms, and a light intensity of 1mW / mm². 2 Five light pulses.

[0046] Technical Effects: This method, through appropriate 470nm blue light irradiation, induces intracellular calcium transients in pacemaker cells within organoids, increasing calcium ion concentration. This calcium signal is transmitted via gap junctions established between early neural cells, forming a calcium wave similar to that in the developing retina. Downstream cytoskeletal protein rearrangement promotes the migration of retinal progenitor cells to the apex of the developing neuroepithelium. Because the development of retinal organoids differs from that of the in vivo retina, lacking vascular support, more progenitor cells must migrate to the apex of the neuroepithelium to access the nutrient-rich photoreceptor differentiation medium. Furthermore, after migration, contact inhibition is relieved, leading to increased progenitor cell proliferation. This simultaneously promotes differentiation and proliferation, resulting in more retinal photoreceptor cells and thus improving the differentiation and culture outcomes of retinal organoids.

[0047] Preferably, as an improvement, this solution also provides a retinal organoid prepared by the above method.

[0048] Technical effects: This scheme uses ChR2 and GCaMP5G double transfection to construct pacemaker cells to prepare retinal organoids, which facilitates rapid activation of calcium waves in the simulated retina, promotes the differentiation of progenitor cells, and makes the retinal layering more realistic, thereby improving the data reliability of its use as a material for research and promoting the development of related disciplines.

[0049] Preferably, as an improvement, this solution also provides an application of retinal organoids, including the application of the aforementioned retinal organoids in regenerative medicine and drug screening.

[0050] Technical effect: The above settings in this scheme facilitate the acquisition of mature and highly consistent retinal organoids. Attached Figure Description

[0051] Figure 1 The following is a comparison of different transfection results during the fluorescence-activated flow cytometry cell sorting process in this embodiment of the invention (A is the viral expression cassette of LV-CAG-hChR2(H134R)-mCherry-WPRE and pLOV-CMV-GCaMP5G; B is the flow cytometry sorting result; C is the cell fluorescence image of different transfection results, from left to right: successful transfection of ChR2, successful transfection of GCaMP5G, and double transfection of ChR2 and GCaMP5G).

[0052] Figure 2 The diagram shows the differentiation and culture process of an embodiment of the present invention (A shows the induced differentiation scheme of retinal organoids; B shows that the induced differentiated retinal organoids have a neuroepithelial layer and express GCaMP5G and mCherry fluorescence; C shows that Arrestin is expressed in retinal organoids, indicating that photoreceptor differentiation was successful).

[0053] Figure 3 The images show the formation and spread of calcium waves in retinal organoids after light stimulation according to an embodiment of the present invention (AC shows the corresponding pseudo-color time-lapse images, ΔF / F0 images, and kymographs of spontaneous calcium waves in retinal organoids after light stimulation, respectively).

[0054] Figure 4 The following are diagrams illustrating the formation and propagation of calcium waves in retinal organoids after photostimulation according to embodiments of the present invention: (A shows a pseudo-color image of calcium waves formed by photostimulation in the neuroepithelial layer; B shows the spatial propagation of calcium signals induced by photostimulation; C shows the spatial propagation of calcium signals induced by photostimulation—cells far from the stimulation area (blue circles) show delayed calcium transients compared to cells near the stimulation area (red and green circles); D shows the photostimulation pattern).

[0055] Figure 5 The diagram illustrates the effect of light stimulation on retinal development and maturation in this invention. (A shows the distribution of cone cells expressing Arrestin before and after light exposure; B shows the distribution of rod cells expressing Nrl before and after light exposure; C and D show the distribution of Arrestin before and after light exposure.) Figure 3 A statistical graph showing the changes in the number of Arr3 (abbreviated as Arr3 in C) and Nrl.

[0056] Figure 6 The diagram shows the changes in retinal calcium waves after 50 hours of light stimulation according to an embodiment of the present invention (AC shows the corresponding pseudo-color time-lapse diagram, ΔF / F0 diagram, and kymograph of calcium waves generated by retinal organoids after 50 hours of light stimulation; D shows the statistical graph of the peak value changes of calcium waves generated before and after 50 hours of light stimulation; EF shows the cumulative frequency histogram of fluorescence response rise and decay dynamics).

[0057] Figure 7 The diagram shows the effect of MOI on fluorescence sorting and organoid differentiation and development in this embodiment of the invention (AC shows the flow cytometry sorting results for MOI=1, 5, and 10, respectively; DF shows the retinal organoids developed after cell sorting for MOI=1-5, 5-10, and 10-20, respectively; G shows the thickness statistics of the neural retina generated after cell sorting for different MOIs).

[0058] Figure 8 The following diagram illustrates the effects of different blue light stimulation intensities on calcium signaling and cell survival in embodiments of the present invention (AC shows 0.1, 1, and 10 mW / mm²). 2 (The staining of Caspase 3 after light intensity irradiation). Detailed Implementation

[0059] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0060] Overview of the Plan

[0061] This protocol provides a method for constructing pacemaker cells and preparing retinal organoids based on ChR2 and GCaMP5G double transfection of hESCs, including the following steps:

[0062] Step 1, Transfection: Transfect ChR2 and GCaMP5G into hESC; ChR2 refers to human ChR2, i.e., hChR2(H134R), the nucleotide sequence of hChR2(H134R) is shown in SEQ_ID_NO.1; the nucleotide sequence of GCaMP5G is shown in SEQ_ID_NO.2; including the following steps:

[0063] A1. Use TrypLE™ Express (Gibco) to isolate hESCs into single cells;

[0064] A2. Suspend in E8 medium containing lentivirus LV-CAG-hChR2(H134R)-mCherry-WPRE (transfection fold can be selected from 5 to 10 times), Y-27632 (content can be selected from 10 to 15 μM) and polyethylene (dosage can be selected from 10 to 15 μg / ml) to prepare a transfection suspension;

[0065] A3. Plate the transfection suspension onto the surface of the vitronectin coating and incubate for 16-18 hours;

[0066] A4. Discard the virus supernatant, add fresh E8 medium containing Y-27632, and incubate for 20-24 hours before removing.

[0067] A5. Repeat steps A2 to A4 to transfect pLOV-CMV-GCaMP5G (the transfection fold can be selected from 5 to 10 times) to obtain a mixed cell solution containing ChR2 and GCaMP5G double transfection.

[0068] Step 2, Screening: Screening for ChR2 and GCaMP5G double-positive cells using FASC; including the following:

[0069] B1. Use papain to dissociate the above mixed cell fluid into single cells;

[0070] B2. After filtration, the dissociated single cells were resuspended in PBS buffer (containing 0.5% FBS and 5 mM EDTA) to form a single-cell suspension.

[0071] B3. The above single-cell suspension was sorted using fluorescently activated cell sorting to obtain ChR2 and GCaMP5G double-positive cell suspensions.

[0072] Step 3: Differentiation and Culture: Two-dimensional ChR2 and GCaMP5G double-positive hESCs are differentiated and cultured into three-dimensional retinal organoids; this includes the following steps:

[0073] C1. The double-positive cell suspension obtained in B3 was dissociated into a single-cell suspension. After centrifugation, the cell pellet was resuspended in retinal differentiation medium containing DNase I and Y-27632 to obtain a cell suspension.

[0074] C2. On day 0, add the cell suspension from C1 to each well of a V-bottom 96-well plate with low cell adhesion for incubation.

[0075] C3. On day 6, the culture medium was replaced with fresh retinal differentiation medium containing 1.5 nM BMP4 (Bone_Morphogenetic_Protein 4) and cultured for another time. Half of the culture medium was replaced every three days to obtain the embryoid body hERO.

[0076] C4. On day 18, the hERO obtained from C3 culture was transferred to a culture dish and cultured in a long-term culture medium until the neural retinal structure appeared, thus obtaining retinal organoids (ROs).

[0077] C5. The ROs obtained in C4 were further induced to differentiate in a long-term induction medium for 70 days.

[0078] The retinal differentiation medium consisted of 45 portions of IMDM medium, 45 portions of F12-Glutamax (F12 medium supplemented with L-alanyl-L-glutamine dipeptide), 1 portion of chemically defined lipid concentrate, 10 portions of serum substitute, 450 μM monothioglycerol, 100 U / ml penicillin, and 100 mg / ml streptomycin. The retinal differentiation medium also contained 0.05 mg / ml DNase I and 20 μM Y-27632. The long-term medium consisted of DMEM / F12-Glutamax medium supplemented with 1% N2, 10% fetal bovine serum, 0.5 μM RA (retinoic acid), 0.1 mM taurine, and 0.25 μg / ml Fungizone (amphotericidal B).

[0079] Step 4: Photostimulation: ChR2 was activated with 470 nm blue light, and calcium signals were observed under a two-photon microscope. Specifically, this included the following: At 70 days of differentiation culture, the cultured ROs were continuously stimulated with 470 nm blue light for 50 hours at a frequency of once every 25 seconds. Each stimulation consisted of a frequency of 100 Hz, a duration of 3 ms, and a light intensity of 1 mW / mm². 2 Five light pulses.

[0080] This scheme also provides a retinal organoid, prepared according to the above method.

[0081] This solution also provides an application of retinal organoids, including their use in regenerative medicine and drug screening.

[0082] Example

[0083] This protocol provides a method for constructing pacemaker cells and preparing retinal organoids based on ChR2 and GCaMP5G double transfection of hESCs, including the following steps:

[0084] 1.1 Transfection

[0085] Human embryonic stem cells (HESCs) were infected with lentiviruses encoding hChR2 (H134R) and GCaMP6s for lentiviral transduction. hESCs (specifically, the human embryonic stem cell line Q-CTS-hESC-2 donated by Professor Hao Jie of the Institute of Zoology, Chinese Academy of Sciences; details can be found in the "Genetic Resource Source Disclosure Registration Form") were isolated into single cells using TrypLE™_Express (Gibco) and then suspended in Essential 8™ medium containing lentivirus LV-CAG-hChR2(H134R)-mCherry-WPRE (purchased from BrainVTA_Technology, infection fold of 5), Y-27632 (purchased from Sigma, 10 μM), and polyethylene (10 μg / ml). The cells were then coated with a vitronectin-coated surface. After 16 hours, the viral supernatant was discarded, and fresh Essential_8™ medium containing Y-27632 was added. The cells were harvested the following day.

[0086] For the control group expressing only hChR2(H134R), infection with LV-CAG-hChR2(H134R)-mCherry-WPRE was performed. One week after mCherry transfection, the control group was transfected with pLOV-CMV-GCaMP5G (purchased from OBiO_Technology, infection fold of 5) to obtain the experimental group, which expressed both hChR2(H134R) and GCaMP5G.

[0087] 1.2 Fluorescence-activated flow cytometry cell sorting

[0088] The transfected hESCs were dissociated into single cells using papain. The dissociated cells were passed through a 30µm filter (Miltenyi, Bergisch-Gladbach, Germany), and resuspended in PBS with 0.5% FBS and 5 mM EDTA to obtain a single-cell suspension (2 × 10⁻⁶ cells / year). 6Single-cell suspensions were analyzed (cells / ml), with unlabeled cells as a control. Flow cytometry was performed on BD_FACS_Aria_II and BD_FACS_Calibur flow cytometers, and data were analyzed using FlowJo software. Results are as follows: Figure 1 As shown. Among them, Figure 1 A shows the viral expression cassettes of LV-CAG-hChR2(H134R)-mCherry-WPRE and pLOV-CMV-GCaMP5G; Figure 1 B displays the flow sorting results; Figure 1 C shows the cell fluorescence images of the transfection results (from left to right: ChR2 channel, GCaMP5G channel, and a combined fluorescence image of ChR2 and GCaMP5G double transfection). The data indicate that flow cytometry successfully screened out ChR2 and GCaMP5G double-positive cells.

[0089] 1.3 Organoid Differentiation Culture:

[0090] New embryonic stem cell lines formed after two sequential viral infections were induced into retinal organoids through multi-step culture.

[0091] Differentiation was performed according to the described method [generating ciliary limb-like stem cell niches from self-organized human retinal tissue]. Briefly, hESCs were broken down into single-cell suspensions using TrypLE_Express (Gibco), the cell pellet was collected by centrifugation at 180g for 2 min, and resuspended in 1 ml of retinal organoid (RO) differentiation medium. This medium contained 0.05 mg / ml DNase_I (Roche) and 20 μM Y-27632 (Merck Cas: 146986-50-7). The retinal differentiation medium consisted of 45% IMDM (Gibco), 45% F12-Glutamax (Gibco), 450 μM monothioglycerol (Sigma-Aldrich), and 1% chemically defined lipid concentrate (Gibco), along with 10% serum substitute (KSR, Gibco) and 100 U / ml penicillin and 100 mg / ml streptomycin (Gibco). On day 0, add 100 μl of a solution containing 1.5 × 10⁻⁶ mg / mL to each well. 4Cell suspensions of 10 cells were transferred to low-cell-adhesion 96-well plates (Sumitomo Bakelite). On day 6, the medium was replaced with fresh retinal differentiation medium containing 1.5 nM bone morphogenetic protein 4 (BMP4, Peprotech). Subsequently, half of the medium was replaced every three days. On day 18, hERO cells were transferred to 9 cm culture dishes (ultra-low adhesion, Corning) and cultured in long-term induction medium consisting of DMEM / F12-Glutamax medium (Gibco) supplemented with 1% N2 (Gibco), 10% fetal bovine serum (FBS, Gibco), 0.5 μM RA (Sigma), 0.1 mM taurine (Sigma), and 0.25 μg / ml Fungizone (Gibco) until the neuroretinal (NR) structure appeared (25-30 days). ROs continued to differentiate using long-term induction medium (LTCM) until day 70. Changes during differentiation culture were as follows. Figure 2 As shown. Among them, Figure 2 A demonstrates the induction and differentiation protocol for retinal organoids; Figure 2 B shows that the induced differentiated retinal organoids have a neuroepithelial layer and express GCaMP5G and mCherry fluorescence; Figure 2 C demonstrates the expression of Arrestin in retinal organoids, indicating successful photoreceptor differentiation. Data show that hESCs transfected with the virus can be successfully induced to differentiate into retinal organoids.

[0092] 1.4 Blue light controls pacemaker cells to initiate retinal wave modulation, organoid stratification, and photoreceptor development.

[0093] Stimulation with 470 nm blue light began on day 70. Stimulation lasted for 50 hours, with a frequency of once every 25 seconds. Each stimulation consisted of five light pulses at a frequency of 100 Hz and a duration of 3 ms. The formation and spread of calcium waves in retinal organoids were observed after stimulation; results are detailed below. Figure 3 and Figure 4 .in, Figure 3 AC shows the corresponding pseudo-color time-lapse images, ΔF / F0 images, and kymographs of spontaneous calcium waves in retinal organoids after light stimulation. Figure 4 A shows a pseudo-color image of calcium waves generated by light stimulation in the neuroepithelial layer; Figure 4 B illustrates the spatial propagation of light-stimulated calcium signals; Figure 4 C illustrates the spatial propagation of light-stimulated calcium signals—cells farther from the stimulation region (blue circles) show delayed calcium transients compared to cells closer to the stimulation (red and green circles); Figure 4D illustrates the light stimulation pattern. Data show that light stimulation can activate pacemaker cells expressing ChR2, and these pacemaker cells can propagate calcium signals to form calcium waves, which are synchronous and propagable.

[0094] Experimental Example 1: Analysis of the developmental maturity of retinal organoids

[0095] The maturity of retinal organoids was determined using an immunofluorescence counting method, with the following steps:

[0096] hROs were fixed in 4% formaldehyde at 4°C for 15–30 minutes, equilibrated overnight in 30% sucrose at 4°C, embedded in OCT (Biosharp), and frozen at -20°C. Sections were then cryogenically prepared to a thickness of 14 μm using a Leica CM1900UV cryostat. At room temperature, sections were blocked with PBS containing 0.1% Triton X-100, 10% FBS, and 1% bovine serum albumin (BSA) for 30–60 minutes. Primary antibody was incubated overnight at 4°C, followed by secondary antibody administration at room temperature for 1–2 hours. Cell nuclei were counterstained with DAPI (1:10, Beyotime) for 10 minutes, then fixed with anti-fading fixative Beyotime, and finally mounted with nail polish.

[0097] The results are as follows Figure 5 As shown. Among them, Figure 5 A shows the distribution of cone cells expressing Arrestin before and after light exposure; Figure 5 B shows the distribution of rod cells expressing Nrl before and after illumination; Figure 5 CD shows statistical graphs illustrating the changes in the number of Arrestin (abbreviated as Arr3 in the figure) and Nrl before and after light exposure. The results indicate that after light stimulation, the fluorescent protein markers Arrestin (cone cells) and Nrl (rod cells), which represent retinal photoreceptors, were more abundant than in the control group, and their orderly arrangement in the outer layer of the neuroepithelium was similar to that in the in vivo outer nuclear layer of the retina.

[0098] Experimental Example 2: Analysis of the functional developmental maturity of retinal organoids

[0099] The developmental maturity of retinal organoids was assessed using a photostimulated two-photon imaging method, following these steps: Real-time calcium imaging of retinal organoids was performed using a two-photon microscope. During imaging, the organoids were held in differentiation culture medium at room temperature. A sutter microscope with movable objectives, tuned to 915 nm (coherent) and an Olympus_LUMPlanFI_40× water immersion objective (NA 0.8), was used. Fluorescence signals were captured using an HQ 535 / 50GFP emission filter (ChROsma_Technology) and processed using Pho_Imagev.3.0 software. Image sequences were acquired at 1 ms per line with a resolution of 256 × 256 pixels and analyzed using Igor_Pro_v.6.10 or ImageJv.1.53t (NIH). Regions of interest were determined based on the standard deviation of the image sequences. Fluorescence intensity (ΔF / F) is calculated as ΔF / F = (F-F0) / F0, where F represents the current GCaMP5G fluorescence and F0 represents the baseline fluorescence.

[0100] The results are as follows Figure 6 As shown. Among them, Figure 6 AC shows the corresponding pseudo-color time-lapse image, ΔF / F0 image, and kymograph of calcium waves generated by retinal organoids after 50 hours of light stimulation. Figure 6 D shows a statistical graph illustrating the peak value changes of calcium waves generated before and after 50 hours of light stimulation; Figure 6 EF shows the cumulative frequency histogram of the fluorescence response rise and fall dynamics. The results indicate that the calcium waves formed by restimulation 50 hours after light stimulation were more uniform and had higher peak values ​​than those formed by the initial stimulation, suggesting that the retinal neural circuits formed by retinal stimulation were more active and that neuronal connectivity was increased, indicating that the retinal organoids were more mature.

[0101] Experiment Example 3: Viral Infection Titer Test

[0102] The effect of MOI on the development of retinal organoids was determined by measuring the cultured organoids after flow cytometry sorting.

[0103] Flow cytometry results under different MOI conditions are as follows Figure 7 As shown, Figure 7 AC displays the streaming sorting results for MOI=1, 5, and 10 respectively. Figure 7 DF shows retinal organoids developed after cell sorting at MOI=1-5, 5-10, and 10-20, respectively. Figure 7G shows a statistical graph of the thickness of the neural retina generated after cell sorting at different MOIs. The results show that: too low an MOI results in low transfection rate and fewer sorted cells; too high an MOI results in low cell activity and a thinner neuroendothelial layer in the generated retinal organoids; therefore, an MOI value of 5 was finally selected.

[0104] Experiment Example 4: Blue Light Stimulation Intensity Test

[0105] Using 0.1, 1, and 10 mW / mm 2 Different light intensities were used to stimulate organoids, and changes in calcium signal were observed using two-photon microscopy. Caspase-3 staining was used to observe cell apoptosis. The results are as follows: Figure 8 As shown. Figure 8 AC values ​​are displayed as 0.1, 1, and 10 mW / mm. 2 The staining of Caspase 3 after light intensity irradiation. Data show that at 10 mW / mm 2 The intensity of light may lead to increased cell apoptosis due to excessive activation of calcium signals, resulting in impaired organoid development.

[0106] In summary, this protocol, through the construction of a dual-virus-infected embryonic stem cell line, ensures that the photogenic cation channel protein (channel_rhodopsin 2, ChR2) and the gene-edited calcium fluorescent indicator protein (GCaMP5G) function normally in both the embryonic stem cells and their differentiated progeny cells. Furthermore, the embryonic stem cells maintain their pluripotency after two transfections and can differentiate into retinal organoids. Upon irradiation with appropriately programmed 470nm blue light, the differentiated retinal organoids induce intracellular calcium transients in pacemaker cells, leading to increased calcium ion concentration. This calcium signal is transmitted through gap junctions established between early neurons, forming a calcium wave similar to that in the developing retina. Downstream cytoskeletal protein rearrangement promotes the migration of retinal progenitor cells to the apex of the developing neuroepithelium. Because the development of retinal organoids differs from that of the in vivo retina, lacking vascular support, more progenitor cells must migrate to the apex of the neuroepithelium to access the nutrient-rich photoreceptor differentiation medium. Simultaneously, after migration, contact inhibition is relieved, resulting in greater progenitor cell proliferation. This achieves the effect of simultaneously promoting differentiation and proliferation, resulting in more retinal photoreceptor cells.

[0107] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing retinal organoids from pacing cells based on ChR2 and GCaMP5G double-transfected hESCs, characterized by: Comprising the following steps: Step one, transfection: transfect ChR2 and GCaMP5G in hESC; Step two, screening: screen ChR2 and GCaMP5G double positive cells by FACS; Step three, differentiation culture: differentiate and culture two-dimensional ChR2 and GCaMP5G double positive hESC into three-dimensional retinal organoids; the differentiation culture comprises the following steps: C1, dissociate the double positive cell suspension into a single cell suspension, centrifuge, and resuspend the cell pellet in a retinal differentiation medium containing DNase_I and Y-27632 to obtain a cell suspension; C2, on day 0, add the cell suspension in C1 to each well of a low cell adhesion v-bottom 96-well plate for culture; C3, on day 6, continue to culture after replacing the culture medium with fresh retinal differentiation medium containing 1.5 nM of BMP4, and replace half of the culture medium every three days, and continue to culture to obtain a hERO; C4, on day 18, move the hERO obtained by culturing in C3 to a culture dish, continue to culture in long-term culture medium until the neural retinal structure appears, and obtain retinal organoids ROs; C5, continue to induce differentiation of the ROs obtained in C4 in the long-term induction medium to 70 days; Step four, light stimulation: At 70 days of differentiation culture, the obtained ROs were continuously stimulated with 470 nm blue light for 50 h, with a frequency of 1 time per 25 seconds, and each stimulation included 5 light pulses with a frequency of 100 HZ, a duration of 3 ms, and a light intensity of 1 mW / mm 2 .

2. The method for preparing retinal organoids from ChR2 and GCaMP5G double transfected hESCs according to claim 1, characterized in that: In step one, LV-CAG-hChR2(H134R)-mCherry-WPRE is used to transfect ChR2, and pLOV-CMV-GCaMP5 is used to transfect GCaMP5G, and the transfection multiples of LV-CAG-hChR2(H134R)-mCherry-WPRE and pLOV-CMV-GCaMP5G are both 5-10 times.

3. The method for preparing retinal organoids from ChR2 and GCaMP5G double transfected hESCs according to claim 1, characterized in that: In step two, the screening comprises the following contents: B1, dissociate the above mixed cell solution into single cells using papain; B2, resuspend the single cells in PBS buffer after filtering the single cells in B1 to obtain a single cell suspension; B3, use fluorescence activated cell sorting to obtain a ChR2 and GCaMP5G double positive cell suspension.

4. The method for preparing retinal organoids from ChR2 and GCaMP5G double transfected hESCs according to claim 1, characterized in that: The retinal differentiation medium is composed of 45 parts of IMDM medium, 45 parts of F12-Glutamax medium, 1 part of chemically defined lipid concentrate, 10 parts of serum substitute, 450 μM monothioglycerol, 100 U / ml penicillin and 100 mg / ml streptomycin; the retinal differentiation medium contains 0.05 mg / ml of DNase_I and 20 μM of Y-27632; the long-term culture medium is composed of DMEM / F12-Glutamax medium, and the medium is added with 1% of N2 supplement, 10% fetal bovine serum, 0.5 μM of RA, 0.1 mM taurine and 0.25 μg / ml of Fungizone.

5. A retinal organoid prepared according to the method of any one of claims 1-4.

6. Use of the retinal organoid prepared according to the method of any one of claims 1-4 in drug screening.

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