Method for preparing retinal organoid by constructing pace-making cells based on ChR2 and GCaMP5G double transfected hESC

Abstract

The invention relates to the technical field of preparation of organoids, and discloses a method for preparing retinal organoids by constructing pace-making cells on the basis of double transfection of hESC (human ESC) by ChR2 and GCaMP5G, which comprises the following steps: step 1, transfection: transfecting ChR2 and GCaMP5G in the hESC; step 2, screening: screening double positive cells of ChR2 and GCaMP5G through FASC (Fibroblast Amplification Specific Cells); step 3, performing differentiation culture: performing differentiation culture on the two-dimensional ChR2 and GCaMP5G double-positive hESC to obtain a three-dimensional retina organ; and 4, photostimulation: activating ChR2 by using 470nm blue light, and observing a calcium signal under a two-photon microscope. According to the scheme, the ChR2 and the GCaMP5G are transfected in the hESC, the double-positive cells of the ChR2 and the GCaMP5G are screened and then cultured into the retina organoid, and the organoid is stimulated by specific blue light, so that the speed of differentiating and culturing the hESC into the three-dimensional retina organoid is effectively increased, the preparation speed of the retina organoid is effectively increased, and the period is shortened.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organoid preparation, and particularly relates to a method for preparing retinal organoids based on ChR2 and GCaMP5G double-transfected hESC. BACKGROUND

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

[0003] Currently, retinal organoids are usually prepared by the following steps: Step one, stem cell culture: embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) are used as starting materials. Step two, directional differentiation: by adding specific growth factors and small molecule compounds, the stem cells are directionally differentiated into retinal precursor cells. Step three, three-dimensional culture: the retinal precursor cells are transferred to a three-dimensional culture system to promote their self-organization into retinal organoids. Step four, maturation culture: after weeks to months of culture, the organoids gradually develop into layered structures similar to the retina, including photoreceptor cells (rods and cones), bipolar cells, ganglion cells, etc. However, the existing technology still has the following defects: (1) The preparation of existing retinal organoids requires multiple steps of differentiation, three-dimensional culture, and long-term maturation, and the entire process usually takes several months (such as 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 Cone Photoreceptors. Stem Cell Reports, 9: 1-18. Available from DOI: 10.1016 / j.stemcr.2017.07.022;recorded 17 weeks), the steps are complicated and technically demanding; and time-consuming, high cost, limiting its application in high-throughput screening or large-scale research. (2) The maturity of the existing retinal organoids is insufficient, although the retinal organoids can simulate the layered structure of the retina, but its maturity is still different from the real retina, especially the function of photoreceptor cells and ganglion cells has not yet fully matured; and the imperfect function of the 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 existing methods may have significant differences in cell composition, structure and function, which significantly reduces the repeatability of the experiment and increases the difficulty of data interpretation. (4) Although the retinal organoids prepared by the existing method can generate photoreceptor cells, the light sensitivity and electrophysiological function of these cells are usually weak, which is difficult to completely simulate the light signal conversion process of the real retina, thereby affecting the results of optogenetic experiments or drug screening.

[0004] Therefore, it is necessary to develop a method for preparing retinal organoids with high success rate, short cycle, consistent cell maturity and good sensitivity, which not only effectively makes up for the shortcomings of the existing technology, but also effectively improves the preparation efficiency and effect of retinal organoids, and has important significance for promoting related experimental research based on retinal organoids. SUMMARY

[0005] The present application aims to provide a method for preparing retinal organoids based on ChR2 and GCaMP5G double-transfected hESC to construct pacemaker cells, to solve the technical problem of long cycle of the existing method for preparing retinal organoids.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: a method for preparing retinal organoids based on ChR2 and GCaMP5G double-transfected hESC to construct pacemaker cells, comprising the following steps: Step one, transfection: transfect ChR2 and GCaMP5G in hESC; Step two, screening: screen ChR2 and GCaMP5G double-positive cells by FASC; Step three, differentiation culture: differentiate and culture two-dimensional ChR2 and GCaMP5G double-positive hESC into three-dimensional retinal organoids; Step four, light stimulation: activate ChR2 with 470nm blue light, and observe calcium signal under a two-photon microscope.

[0007] The principle of the present application is as follows: ChR2 (channelrhodopsin-2, a light-sensitive cation channel) is a light-controlled cation channel, which opens upon activation, and the inward flow of cations causes the membrane potential to depolarize, and is conducted to other cells through gap junctions, eventually forming calcium waves similar to those observed in developing retinas. GCaMP5G is a genetically edited calcium fluorescence indicator, which is composed of green fluorescent protein (GFP), calmodulin (CaM) and a peptide segment M13 of myosin light chain kinase. When ChR2 is activated, calcium ions flow in and bind to CaM, inducing a conformational change in CaM, which binds to the light chain region of M13, thereby enhancing the degree of protonation of the GFP chromophore under excitation at a specific wavelength, resulting in an increase in absorbance and a significant increase in fluorescence intensity, thereby realizing the visualization of ChR2-mediated calcium waves. The present scheme transfects ChR2 in hESC, and the successfully transfected ChR2 positive cells have the characteristics of pacemaker cells, which are the key role in the whole calcium wave formation process. By activating the open channel, it drives other cells to excite after initial excitation, effectively simulating the calcium wave in the retina, thereby promoting the migration and differentiation of progenitor cells, making the retinal stratification more realistic, and more PR (Photoreceptor, photoreceptor) survive in the normal neuroepithelial outer layer to complete the functional development, and more neurons form more active retinal neural circuits, thereby further promoting the maturation of the whole organoid.

[0008] The advantages of the present scheme are: 1. Compared with the long period of preparing retinal organoids in the prior art, the present scheme transfects ChR2 and GCaMP5G in hESC, and then cultures after screening ChR2 and GCaMP5G double positive cells, which effectively speeds up the differentiation of hESC into three-dimensional retinal organoids, thereby effectively improving the preparation speed of retinal organoids and shortening the period.

[0009] 2. Compared with the large batch-to-batch difference of the prepared retinal organoids in the prior art, the present scheme can mass-produce retinal organoids by transfection, screening, differentiation culture and light stimulation, and the structure and function of the obtained retinal organoids have high similarity and small batch-to-batch difference, thereby improving the repeatability of experiments based on retinal organoids, reducing the difficulty of data interpretation, and promoting the rapid development of related disciplines.

[0010] 3. The present scheme uses the optogenetic tool ChR2 to construct pacemaker cells, and induces the excitation of pacemaker cells by light control, thereby generating calcium waves, which has a significant impact on promoting the development of retinal organoids, and has an ideal application prospect.

[0011] 4、This scheme is to transfect ChR2 and GCaMP5G in hESC (human embryonic stem cells) at the same time, so as to detect the effect of differentiation culture and activation quickly after ChR2 is activated by blue light, causing the change of cell membrane potential, triggering calcium ion influx. Specifically, GCaMP5G can combine with calcium ions (Ca 2+ ) in cells, and when the concentration of calcium ions increases, GCaMP5G will emit a fluorescence signal. Therefore, this scheme transfects GCaMP5G into hESC, and in the differentiated retinal organoids, the dynamic changes of calcium signals in the retinal organoids can be observed in real time through a two-photon microscope, so as to study the signal transmission and functional activity between cells.

[0012] 5、This scheme is to transfect ChR2 and GCaMP5G in hESC cells at the same time, and to use FASC (fluorescence-activated cell sorting) for screening, so as to quickly screen for double-positive cells and improve efficiency. Specifically, after successful transfection of ChR2, hESC cells will express red fluorescent markers, and after successful transfection of GCaMP5G, hESC cells will express green fluorescent markers. The simultaneous presence of red and green fluorescent markers facilitates the rapid detection, analysis and sorting of cells by FASC according to the fluorescent markers of the cells, thereby realizing the rapid screening of double-positive cells and further improving the efficiency of preparing retinal organoids.

[0013] Preferably, as an improvement, in step one, transfection includes the following steps: A1, separate hESCs into single-cell hESCs; A2, suspend the hESCs in A1 in E8 medium containing lentivirus LV-CAG-hChR2(H134R)-mCherry-WPRE, Y-27632 and polyethylene to prepare a transfection suspension; A3, coat the transfection suspension on the surface of a vitronectin coating and culture for 16-18 h; A4, discard the virus supernatant, add fresh E8 medium containing inhibitor Y-27632, and take out after 20-24 h of culture; A5, repeat steps A2-A4, replace LV-CAG-hChR2(H134R)-mCherry-WPRE in A2 with pLOV-CMV-GCaMP5G to obtain a mixed cell solution containing ChR2 and GCaMP5G double transfection.

[0014] Technical effects: The above-mentioned settings are adopted to improve the success rate of double transfection. Specifically, the selection of lentivirus (such as lentivirus LV-CAG-hChR2(H134R)-mCherry-WPRE and lentivirus pLOV-CMV-GCaMP5G) transfection can integrate the transfection content into the genomic DNA of the target cell, thereby facilitating long-term expression. Y-27632 is a ROCK inhibitor that can reduce cell apoptosis and mechanical damage, and is particularly suitable for single-cell suspension culture or cells after transfection; inhibiting the ROCK signaling pathway can also reduce the adhesion between cells and prevent cell aggregation, thereby improving transfection efficiency. Polyethylene is a cationic polymer that can form a complex with DNA to facilitate DNA entry into cells. Vitronectin is an extracellular matrix protein that contains an RGD (arginine-glycine-aspartic acid) sequence that can bind to integrin receptors on the cell surface to promote cell adhesion. In transfection experiments, Vitronectin coating can improve cell adhesion and survival rate, thereby optimizing transfection efficiency.

[0015] Preferably, as an improvement, the Y-27632 content in the E8 medium is 10-15 μM, and the polyethylene dosage is 10-15 μg / ml.

[0016] Technical effects: The above-mentioned settings are adopted to optimize and improve the survival rate and transfection efficiency of difficult-to-transfect cells (such as stem cells). Specifically, the applicant has found through long-term experiments that if the Y-27632 content is too high, the cells will be too dispersed, affecting cell-to-cell signaling and function. And too much Y-27632 can interfere with the normal physiological functions of cells (such as differentiation ability) and affect transfection efficiency; if the Y-27632 content is too low, the cells may aggregate due to insufficient inhibition of the ROCK signaling pathway, reducing transfection efficiency; and cells may die in large numbers due to mechanical damage or stress during transfection. If the polyethylene dosage is too high, it may be toxic to cells, leading to cell death; and too much polyethylene may result in DNA complexes that are too large to enter cells, reducing transfection efficiency. In addition, excessive polyethylene may non-specifically bind to cell membranes, affecting cell function. If the polyethylene dosage is too low, the DNA complex may not form completely, reducing transfection efficiency, and may even fail to effectively protect DNA from nuclease degradation.

[0017] Preferably, as an improvement, the transfection multiples of LV-CAG-hChR2(H134R)-mCherry-WPRE and pLOV-CMV-GCaMP5G are both 5-10 times.

[0018] Technical effects: The above-mentioned settings facilitate consideration of transfection efficiency and cell activity after transfection. The applicant has found through long-term experiments that too low MOI (transfection multiple) will result in low transfection rate, difficulty in building a strain, or weak light gene function of infected cells, which is difficult to detect; and too high MOI will result in loss of cell pluripotency, difficulty in proliferation and survival, or failure of organoid differentiation.

[0019] Preferably, as an improvement, in step two, the screening includes the following: B1, dissociating the above-mentioned mixed cell solution into single cells using papain; B2, resuspending the single cells in B1 into a single cell suspension with PBS buffer; B3, obtaining a ChR2 and GCaMP5G double-positive cell suspension by fluorescence-activated cell sorting of the above-mentioned single cell suspension.

[0020] Technical effects: The above-mentioned settings facilitate rapid screening of ChR2 and GCaMP5G double-positive cell suspension. Specifically, the present application first dissociates the single cell suspension by papain to maintain cell activity. Then, by filtering and resuspending in PBS (phosphate buffered saline), the high-quality single cell suspension is prepared by removing the lumps and fragments. Finally, by FACS sorting, a high-purity ChR2 and GCaMP5G double-positive cell suspension is obtained for subsequent experiments.

[0021] Preferably, as an improvement, in step three, the differentiation culture includes the following steps: C1, dissociating the double-positive cell suspension obtained in B3 into a single cell suspension, centrifuging, and resuspending the cell pellet in a retinal differentiation medium containing DNase I and Y-27632 to obtain a cell suspension; C2, on day 0, adding the cell suspension in C1 to each well of a low-cell-adhesion v-bottom 96-well plate for culture; C3, on day 6, replacing the culture medium with fresh retinal differentiation medium containing 1.5 nM of BMP4 and continuing to culture, and replacing half of the culture medium every three days, to obtain a hERO; C4, on day 18, moving the hERO obtained by culturing in C3 to a culture dish and continuing to culture in long-term culture medium until the appearance of neural retinal structures, to obtain retinal organoids ROs; C5, continuing to induce differentiation of the ROs obtained in C4 in long-term induction medium to 70 days.

[0022] Technical effects: The above-mentioned settings facilitate rapid differentiation and culture of double-transfected cells into mature retinal organoids.

[0023] Preferably, as an improvement, 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 replacement, 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 culture medium is composed of DMEM / F12-Glutamax medium, to which 1% N2 supplement, 10% fetal bovine serum, 0.5 μM RA, 0.1 mM taurine and 0.25 μg / ml Fungizone are added.

[0024] Technical effects: The above settings are adopted in the scheme. The retinal differentiation medium is used to induce differentiation of pluripotent stem cells into retinal cells and form retinal organoids. The long-term culture medium is used to maintain long-term culture of the retinal organoids and promote maturation and functional maintenance thereof. The applicant has found through long-term experiments that if the content of Y-27632 in the retinal differentiation medium is too low, directional differentiation of retinal progenitor cells will be blocked, resulting in reduced production of photoreceptors or damaged structure. If the use amount of penicillin and streptomycin is too low, the bacteriostatic ability will be insufficient, resulting in cell death and abnormal differentiation.

[0025] Preferably, as an improvement, in step four, the light stimulation includes the following contents: when the differentiation culture is performed for 70 days, the obtained ROs are continuously stimulated with 470 nm blue light for 50 h, at a frequency of 1 time per 25 seconds. Each stimulation includes 5 light pulses with a frequency of 100 HZ, a duration of 3 ms and a light intensity of 1 mW / mm 2 .

[0026] Technical effects: After irradiation with 470 nm blue light with a suitable program, intracellular calcium transients are generated in the pacemaker cells in the organoids. The concentration of calcium ions is increased, and the calcium signal is transmitted through the gap junctions established between early neural cells, forming a retinal calcium wave similar to that in the developing retina. Through the rearrangement effect of the downstream cytoskeletal proteins of the calcium signal, the migration of retinal precursor cells to the apical end of the responding developing neuroepithelium is promoted. Since the development process of the retinal organoids is different from that of the in vivo retina, the blood vessel system is lacking, and only the migration of more precursor cells to the apical end of the neuroepithelium makes it possible to contact the nutrient-rich photoreceptor differentiation medium. At the same time, after the migration occurs, the contact inhibition is removed, and the precursor cells proliferate more. This achieves the effect of promoting differentiation and proliferation at the same time, and more retinal photoreceptor cells are obtained, thereby improving the differentiation culture effect of the retinal organoids.

[0027] Preferably, as an improvement, the scheme also provides a retinal organoid prepared by the above method.

[0028] Technical effects: The scheme is based on ChR2 and GCaMP5G double transfection to construct a pacing cell to prepare a retinal organoid, which is convenient for quickly activating calcium waves in a simulated retina, promoting the differentiation of progenitor cells, making the retinal stratification more realistic, improving the data reliability of the material research, and thus promoting the development of related discipline research.

[0029] Preferably, as an improvement, the scheme also provides a retinal organoid, including the application of the above-mentioned retinal organoid in regenerative medicine and drug screening.

[0030] Technical effects: The scheme adopts the above-mentioned setting, which is convenient for obtaining mature and consistent retinal organoids. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 For the comparison chart of different transfection results in the fluorescence activated flow cytometry sorting process in the embodiment of the application (A is the virus expression box of LV-CAG-hChR2(H134R)-mCherry-WPRE and pLOV-CMV-GCaMP5G; B is the flow sorting result; C is the cell fluorescence graph of different transfection results, from left to right, respectively: successful transfection of ChR2, successful transfection of GCaMP5G, double transfection of ChR2 and GCaMP5G).

[0032] Figure 2 For the differentiation culture process chart of the embodiment of the application (A shows the induction differentiation scheme of the retinal organoid; B shows that the induced differentiated retinal organoid has a neural epithelial layer and expresses GCaMP5G and mCherry fluorescence; C shows that Arrestin is expressed in the retinal organoid, indicating that the photoreceptor differentiation is successful).

[0033] Figure 3 For the calcium wave formation and spread chart of the retinal organoid after light stimulation (A-C respectively show the corresponding pseudo-color time-lapse graph, ΔF / F0 graph and kymograph graph of the spontaneous calcium wave in the retinal organoid after light stimulation).

[0034] Figure 4 For the calcium wave formation and spread chart of the retinal organoid after light stimulation (A shows the pseudo-color graph of calcium wave formation in the neural epithelial layer after light stimulation; B shows the spatial propagation of calcium signals induced by light stimulation; C shows the spatial propagation of calcium signals induced by light stimulation - compared with the cells close to the stimulation (red and green circles), the cells far from the stimulation area (blue circles) show delayed calcium transients; D shows the light stimulation mode).

[0035] Figure 5Figure 2 shows the plots of the retinal maturation promoted by the light stimulation of the embodiments of the present application (A shows the distribution of the cone cells expressing Arrestin before and after light exposure; B shows the distribution of the rod cells expressing Nrl before and after light exposure; C-D show the statistical plots of the number changes of Arrestin (abbreviated as Arr3 in C) and Nrl, respectively, before and after light exposure). Figure 3

[0036] Figure 6 Figure 4 shows the plots of the changes of the retinal calcium waves after 50h light stimulation of the embodiments of the present application (A-C show the corresponding pseudo-color time-lapse, ΔF / F0 and kymograph plots of the calcium waves generated by the retinal organoids after 50h light stimulation; D shows the statistical plot of the peak changes of the calcium waves generated before and after 50h light stimulation; E-F show the cumulative frequency histograms of the rising and decay kinetics of the fluorescence responses).

[0037] Figure 7 Figure 5 shows the plots of the effects of MOI on fluorescence sorting and organoid differentiation and development of the embodiments of the present application (A-C show the flow sorting results of MOI = 1, 5, 10, respectively; D-F show the retinal organoids developed from the cells sorted at MOI = 1-5, 5-10, 10-20, respectively; G shows the statistical plot of the thickness of the neural retina generated from the cells sorted at different MOI).

[0038] Figure 8 Figure 6 shows the plots of the effects of different blue light stimulation intensities on calcium signals and cell survival of the embodiments of the present application (A-C show the staining of Caspase3 after 0.1, 1, 10mW / mm 2 light intensity exposure, respectively). DETAILED DESCRIPTION

[0039] The present application will be further described below in conjunction with the embodiments, but the embodiments of the present application are not limited thereto. If not specifically indicated, the technical means used in the following embodiments and experimental examples are the conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial channels.

[0040] General description of the scheme The present scheme provides a method for preparing retinal organoids from pacemaker cells based on ChR2 and GCaMP5G double transfection of hESC, comprising the following steps: Step one, transfection: transfect ChR2 and GCaMP5G in 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; comprising the following steps: ​A1, dissociate hESCs into single cells using TrypLE™ Express (Gibco); A2, suspend in E8 medium containing lentivirus LV-CAG-hChR2(H134R)-mCherry-WPRE (transfection fold can be selected in the range of 5-10 times), Y-27632 (content can be selected in the range of 10-15 μM) and polyethylene (use amount can be selected in the range of 10-15 μg / ml) to prepare a transfection suspension; A3, plate the transfection suspension on the surface of a vitronectin coating and culture for 16-18 h; A4, discard the virus supernatant, add fresh E8 medium containing Y-27632, and take out after 20-24 h of culture; A5, repeat steps A2-A4 to transfect pLOV-CMV-GCaMP5G (transfection fold can be selected in the range of 5-10 times) to obtain a mixed cell solution containing ChR2 and GCaMP5G double transfection.

[0041] Step two, screening: screen ChR2 and GCaMP5G double positive cells by FASC; including the following contents: B1, dissociate the above mixed cell solution into single cells using papain; B2, after filtration, resuspend the dissociated single cells in PBS buffer (containing 0.5% FBS and 5 mM EDTA) to obtain a single cell suspension; B3, sort the above single cell suspension by fluorescence activated cell sorting to obtain a ChR2 and GCaMP5G double positive cell suspension.

[0042] Step three, differentiation culture: differentiate and culture two-dimensional ChR2 and GCaMP5G double positive hESCs into three-dimensional retinal organoids; including the following steps: C1, dissociate the double positive cell suspension obtained in B3 into a single cell suspension, centrifuge, 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, replace the culture medium with fresh retinal differentiation medium containing 1.5 nM of BMP4 (Bone_Morphogenetic_Protein 4, bone morphogenetic protein 4) and continue to culture, and replace half of the medium every three days, to obtain a pseudoembryonic hERO. C4, on day 18, the hEROs obtained from C3 were transferred to culture dishes and continued to be cultured in long-term culture medium until the neural retinal structure appeared, obtaining retinal organoids ROs; C5, the ROs obtained in C4 were continued to be induced and differentiated in long-term induction medium until day 70.

[0043] Wherein, the retinal differentiation medium is composed of 45 parts of IMDM medium, 45 parts of F12-Glutamax (F12 medium added with L-alanyl-L-glutamine dipeptide), 1 part of chemically defined lipid concentrate, 10 parts of serum replacement, 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 culture medium is composed of DMEM / F12-Glutamax medium, and the medium is added with 1% N2 supplement, 10% fetal bovine serum, 0.5 μM RA (retinoic acid), 0.1 mM taurine and 0.25 μg / ml Fungizone (amphotericin B).

[0044] Step four, light stimulation: ChR2 is activated with 470 nm blue light, and calcium signals are observed under a two-photon microscope; specifically including the following contents: when the culture obtained ROs are cultured for 70 days, 470 nm blue light is used to continuously stimulate the ROs for 50 h, the frequency is 1 time per 25 seconds, and each stimulation includes 5 light pulses with a frequency of 100 HZ, a time length of 3 ms and a light intensity of 1 mW / mm 2 .

[0045] The scheme also provides a retinal organoid prepared according to the above method.

[0046] The scheme also provides an application of a retinal organoid, including the application of the retinal organoid in regenerative medicine and drug screening.

[0047] Embodiment The scheme provides a method for preparing retinal organoids based on ChR2 and GCaMP5G double transfection of hESC, including the following steps: 1.1 Transfection hESCs (specifically, human embryonic stem cell line (Q-CTS-hESC-2) gifted by Professor Haojie Hu of the Institute of Animal Science, Chinese Academy of Sciences, see "Disclose the registration form of genetic resources source" for relevant information) were infected with LV encoding hChR2 (H134R) and GCaMP6s for lentiviral transduction, and then were dissociated into single cells using TrypLE™_Express (Gibco), and then were suspended in Essential 8TM medium containing lentivirus LV-CAG-hChR2(H134R)-mCherry-WPRE (purchased from BrainVTA_Technology, infection multiple of 5), Y-27632 (purchased from Sigma, 10 μM) and polyethylene (10 μg / ml). The cells were then plated on a vitronectin-coated surface. After 16 h, the viral supernatant was discarded, and fresh Essential_8TM medium containing Y-27632 was added, and the cells were removed the next day.

[0048] For the control group expressing only hChR2 (H134R), LV-CAG-hChR2(H134R)-mCherry-WPRE was used for infection. After 1 week of mCherry transfection, pLOV-CMV-GCaMP5G (purchased from OBiO_Technology, infection multiple of 5) was used for re-transfection to obtain the experimental group, which simultaneously expressed hChR2 (H134R) and GCaMP5G.

[0049] 1.2 Fluorescence-activated cell sorting The aforementioned transfected hESCs were dissociated into single cells with papain, and the dissociated cells were resuspended with 0.5% FBS and 5 mM EDTA in PBS through a 30 µm filter (Miltenyi, Bergisch Gladbach, Germany) to obtain a single cell suspension (2 × 10 6 The single cell suspension was detected with unlabeled cells as a control. Flow cytometry was performed on a BD_FACS_Aria_II and a BD_FACS_Calibur flow cytometer, and the data were analyzed with FlowJo software. The results are shown in Figure 1 Figure 1 A shows the viral expression cassette of LV-CAG-hChR2(H134R)-mCherry-WPRE and pLOV-CMV-GCaMP5G; Figure 1 B shows the results of flow sorting; Figure 1 ​C Cell fluorescence images showing transfection results (from left to right: ChR2 channel, GCaMP5G channel, merged fluorescence images of ChR2 and GCaMP5G double transfection). The data show that ChR2 and GCaMP5G double positive cells are successfully sorted by flow cytometry.

[0050] 1.3 Organoid differentiation culture: The new embryonic stem cell strain formed after two sequential viral infections was induced into retinal organoids through multi-step culture.

[0051] Differentiation was performed as described [Generation of ciliary marginal zone-like stem cell niches from self-organizing human retinal tissue]. Briefly, hESCs were dissociated into single cell suspension using TrypLE_Express (Gibco), cells were collected by centrifugation at 180 g for 2 min, and the cell pellet was resuspended in 1 ml of retinal organoid (RO) differentiation medium. This contained 0.05 mg / ml DNase_I (Roche) and 20 mM Y-27632 (Merck Cas: 146986-50-7). The retinal differentiation medium consisted of 45% IMDM (Gibco), 45% F12-Glutamax (Gibco), 450 mM monothioglycerol (Sigma-Aldrich) and 1% Chemically Defined Lipid Concentrate (Gibco), and 10% serum replacement (KSR, Gibco) and 100 U / ml penicillin and 100 mg / ml streptomycin (Gibco). On day 0, 100 ml of cell suspension containing 1.5 x 10 4 cells were added per well into low cell-adherent v-bottom 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, the medium was replaced every three days with half of the volume. On day 18, hEROs were moved to 9 cm culture dishes (ultra-low attachment, Corning) and cultured in long-term medium consisting of DMEM / F12-Glutamax medium (Gibco) supplemented with 1% N2 (Gibco), 10% fetal bovine serum (FBS, Gibco), 0.5 mM RA (Sigma), 0.1 mM taurine (Sigma) and 0.25 mg / ml Fungizone (Gibco) until the appearance of neural retinal (NR) structures (25-30 days), and ROs continued to be induced to differentiate to 70 days using long-term induction medium (LTCM). Changes during the differentiation culture are shown in Figure 2 Figure 2 ​A Induced differentiation protocol showing retinal organoids Figure 2 B Induced differentiated retinal organoids showing neuroepithelial layer and expression of GCaMP5G and mCherry fluorescence Figure 2 C Arrestin expression in retinal organoids indicating successful photoreceptor differentiation. Data show that hESCs can be successfully induced to differentiate retinal organoids after viral transfection. 1.4 Blue light controlled pacemaker cells initiate retinal wave coordinated organoid stratification and photoreceptor development Stimulation with 470 nm blue light was initiated at day 70. Stimulation lasted 50 h at a frequency of 1 pulse every 25 seconds, each pulse consisting of 5 light pulses at a frequency of 100 HZ for 3 ms. Retinal organoid calcium wave formation, propagation was observed after stimulation, results are shown in Figure 3 and Figure 4 . Among them, Figure 3 A-C show the corresponding false-color time-lapse, ΔF / F0 and kymograph plots of spontaneous calcium waves in retinal organoids after light stimulation; Figure 4 A shows false-color plots of calcium wave formation in the neuroepithelial layer after light stimulation; Figure 4 B shows spatial propagation of calcium signals induced by light stimulation; Figure 4 C shows spatial propagation of calcium signals induced by light stimulation - cells far from the stimulation area (blue circles) show delayed calcium transients compared to cells close to the stimulation (red and green circles); Figure 4 D shows light stimulation pattern. Data show that light stimulation can activate ChR2 expressing pacemaker cells, which propagate calcium signals to form calcium waves, which are synchronous and propagatable.

[0052] Experimental Example 1: Analysis of retinal organoid developmental maturity The developmental maturity of retinal organoids was detected by reference immunofluorescence counting method, the steps are as follows: hROs were fixed in 4% formaldehyde at 4°C for 15 to 30 minutes, balanced in 30% sucrose at 4°C overnight, embedded in OCT (Biosharp), and frozen into shape at -20°C, and 14 μm thick cryosections were performed using Leica CM1900 UV cryostat. At room temperature, block the sections with 0.1% Triton_X-100, 10% FBS and 1% bovine serum albumin (BSA) in PBS for 30-60 minutes. Incubate the primary antibody at 4°C overnight, then apply the secondary antibody at room temperature for 1-2 h. Apply DAPI (1:10, Biyun) for 10 minutes to restain the cell nucleus, then fix with anti-fade mounting medium (Biyun), and finally mount with nail polish.

[0053] The results are shown in Figure 5 . Among them,Figure 5 A shows the distribution of Arrestin-expressing cone cells before and after light stimulation; Figure 5 B shows the distribution of Nrl-expressing rod cells before and after light stimulation; Figure 5 C-D respectively show the statistical graphs of the number changes of Arrestin (abbreviated as Arr3 in the figure) and Nrl before and after light stimulation. The results show that the fluorescence protein markers Arrestin (cone cells) and Nrl (rod cells) representing the photoreceptors of the retina are more than the control group after light stimulation, and are arranged in order in the outer layer of the neuroepithelium and in the outer nuclear layer of the retina in vivo.

[0054] Experimental Example 2: Analysis of the functional development maturity of the retinal organoids The development maturity of the retinal organoids was detected by the method of two-photon imaging of the light-stimulated retinal organoids, and the steps were as follows: real-time calcium imaging of the retinal organoids was performed using a two-photon microscope. During imaging, the organoids were kept in the differentiation medium at room temperature. A movable objective microscope (Sutter) equipped with a chameleon titanium-sapphire laser (coherent) tuned to 915 nm and an Olympus_LUMPlanFI_40x water immersion objective (NA 0.8) was used. The fluorescent signal was captured using a HQ 535 / 50 GFP emission filter (ChROsma_Technology), and processed using Pho_Image v.3.0 software. Image sequences were acquired at a rate of 1 ms per line, with a resolution of 256 x 256 pixels, and analyzed using Igor_Pro_v.6.10 or ImageJ v.1.53t (NIH), with the region of interest determined according to the standard deviation of the image sequence. The fluorescence intensity (ΔF / F) was calculated as ΔF / F = (F-F0) / F0, where F represents the current GCaMP5G fluorescence, and F0 represents the baseline fluorescence.

[0055] The results are shown in Figure 6 , wherein Figure 6 A-C respectively show the corresponding pseudo-color time-lapse, ΔF / F0 and kymograph graphs of the calcium waves generated by re-stimulating the retinal organoids after 50h of light stimulation; Figure 6 D shows the statistical graph of the peak value changes of the calcium waves generated before and after 50h of light stimulation; Figure 6 E-F show the cumulative frequency histograms of the rising and decay kinetics of fluorescence response. The results show that the calcium waves generated by re-stimulation after 50h of light stimulation are more uniform and have higher peak values than the calcium waves generated by initial stimulation, indicating that the retinal neural circuit is more active and the neuronal connectivity is increased, i.e. the retinal organoids are more mature.

[0056] Experimental Example 3: Virus infection titer test The effect of MOI on the development of retinal organoids was detected by culturing organoids after flow sorting.

[0057] The flow sorting results under different MOI conditions are shown in Figure 7 Figure 7 A-C show the flow sorting results of MOI = 1, 5, and 10, respectively. Figure 7 D-F show the retinal organoids developed from cells sorted under MOI = 1-5, 5-10, and 10-20, respectively. Figure 7 G shows the thickness statistics of neural retinas generated from cells sorted under different MOI. The results show that: too low MOI has low transfection rate and few sorted cells; too high MOI has low cell activity and thin neural endothelial layer of generated retinal organoids; therefore, the MOI value of 5 is finally selected.

[0058] Experimental Example 4: Blue light stimulation intensity test Different light intensities of 0.1, 1, and 10 mW / mm 2 were used to stimulate organoids, two-photon microscopy was used to observe calcium signal changes, and Caspase3 staining was used to observe cell apoptosis, and the results are shown in Figure 8 Figure 8 A-C show the Caspase3 staining after irradiation under light intensities of 0.1, 1, and 10 mW / mm 2 respectively. The data show that the light intensity of 10 mW / mm 2 may cause an increase in cell apoptosis due to excessive activation of calcium signals, leading to impaired development of organoids.

[0059] In summary, the present scheme constructs a double-virus-infected embryonic stem cell strain, and both the light gene cation channel protein (channel_rhodopsin 2, ChR2) and the gene-edited calcium fluorescence indicator protein (GCaMP5G) work normally in the embryonic stem cells and their differentiated progeny cells. Moreover, the embryonic stem cells maintain their pluripotency after two transfections and can differentiate into retinal organoids. After irradiation of the retinal organoids obtained by differentiation under a suitable program of 470 nm blue light, the pacemaker cells in the organoids produce intracellular calcium transients, the calcium ion concentration increases, and the calcium signals are transmitted through the gap junctions established between early neural cells, forming a retinal calcium wave similar to that in the developing retina. Through the rearrangement effect of the downstream cytoskeletal proteins of calcium signals, the retinal precursor cells are promoted to migrate to the top of the neural epithelium. Since the development process of retinal organoids is different from that of in vivo retinas, which lacks vascular system support, only the migration of more precursor cells to the top of the neural epithelium can contact the nutrient-rich photoreceptor differentiation medium, and at the same time, the contact inhibition is removed after migration, and the precursor cells proliferate more. In this way, the effects of differentiation and proliferation are promoted at the same time, and more retinal photoreceptor cells are obtained. ​​

[0060] The above is only an embodiment of the present application, and the specific technical solutions and / or common knowledge of the scheme are not described in detail. It should be pointed out that for those skilled in the art, without departing from the technical solutions of the present application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the present application, and these will not affect the effect and practicality of the patent. The protection scope claimed in this application should be subject to the content of its claims, and the specific implementation mode and the like recorded in the specification can be used to explain 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: The method comprises the following steps: Step one, transfection: transfecting ChR2 and GCaMP5G in hESCs; Step two, screening: screening ChR2 and GCaMP5G double positive cells by FACS; Step three, differentiation culture: differentiating and culturing two-dimensional ChR2 and GCaMP5G double positive hESCs into three-dimensional retinal organoids; Step four, light stimulation: activating ChR2 with 470 nm blue light, and observing calcium signals under a two-photon microscope.

2. The method for preparing retinal organoids from ChR2 and GCaMP5G double transfected hESCs according to claim 1, characterized in that: In step one, transfection comprises the following steps: A1, separating hESCs into single-cell hESCs; A2, suspending hESCs in A1 in E8 medium containing lentivirus LV-CAG-hChR2(H134R)-mCherry-WPRE, Y-27632 and polyethylene to prepare a transfection suspension; A3, plating the transfection suspension on a vitronectin-coated surface and culturing for 16-18 hours; A4, discarding the viral supernatant, adding fresh E8 medium containing inhibitor Y-27632, and taking out after culturing for 20-24 hours; A5, repeating steps A2-A4, replacing LV-CAG-hChR2(H134R)-mCherry-WPRE in A2 with pLOV-CMV-GCaMP5G to obtain a mixed cell solution containing ChR2 and GCaMP5G double transfection.

3. The method for preparing retinal organoids from ChR2 and GCaMP5G double transfected hESCs according to claim 2, characterized in that: The content of Y-27632 in the E8 medium is 10-15 μM, and the amount of polyethylene is 10-15 μg / ml.

4. The method for preparing retinal organoids from ChR2 and GCaMP5G double transfected hESCs according to claim 2, wherein: The transfection multiples of LV-CAG-hChR2(H134R)-mCherry-WPRE and pLOV-CMV-GCaMP5G are both 5-10 times.

5. The method for preparing retinal organoids from ChR2 and GCaMP5G double transfected hESCs according to any one of claims 2-4, wherein: In step two, screening comprises the following contents: B1, dissociating the above-mentioned mixed cell solution into single cells using papain; B2, resuspending the single cells in PBS buffer after filtering the single cells in B1 to obtain a single cell suspension; B3, obtaining a ChR2 and GCaMP5G double positive cell suspension by fluorescence activated cell sorting of the above-mentioned single cell suspension.

6. The method for preparing retinal organoids from ChR2 and GCaMP5G double transfected hESCs according to claim 5, wherein: In step three, differentiation culture comprises the following steps: C1, dissociating the double positive cell suspension obtained in B3 into a single cell suspension, centrifuging, resuspending the cell precipitate in a retinal differentiation medium containing DNase_I and Y-27632 to obtain a cell suspension; C2, on day 0, adding the cell suspension in C1 to each well of a low cell adhesion v-bottom 96-well plate for culture; C3, on day 6, replacing the culture medium with fresh retinal differentiation medium containing 1.5 nM of BMP4, continuing to culture, and replacing half of the culture medium every three days, and continuing to culture to obtain a pseudo-embryonic hERO; C4, on day 18, moving the hERO obtained by culturing in C3 to a culture dish, continuing to culture in long-term culture medium until a neural retinal structure appears, and obtaining retinal organoids ROs; C5, continuing to induce differentiation of the ROs obtained in C4 in the long-term induction medium to 70 days.

7. The method for preparing retinal organoids from ChR2 and GCaMP5G double transfected hESCs according to claim 6, characterized in that: The retinal differentiation medium consists of 45 parts of IMDM medium, 45 parts of F12-Glutamax medium, 1 part of chemically defined lipid concentrate, 10 parts of serum replacement, 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 consists of DMEM / F12-Glutamax medium, to which 1% of N2 supplement, 10% fetal bovine serum, 0.5 μM of RA, 0.1 mM taurine and 0.25 μg / ml of Fungizone are added.

8. The method for preparing retinal organoids from ChR2 and GCaMP5G double transfected hESCs according to claim 7, characterized in that: In step four, the light stimulation comprises the following: at 70 days of differentiation culture, the obtained ROs are continuously stimulated with 470 nm blue light for 50 h, at a frequency of 1 time per 25 seconds, and each stimulation comprises 5 light pulses with a frequency of 100 HZ, a duration of 3 ms, and a light intensity of 1 mW / mm 2 .

9. A retinal organoid prepared according to the method of any one of claims 1-8.

10. Use of a retinal organoid produced according to the method of any one of claims 1 to 8, characterized in that: The use of the retinal organoid in drug screening.

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