CN108795864B - Method for obtaining retinal-like tissue rich in cones and rods by using human induced pluripotent stem cells - Google Patents

Abstract

The invention discloses a method for obtaining a retinal-like tissue rich in cones and rods by using human induced pluripotent stem cells. Digesting hiPSCs to obtain cell sediment, and then performing suspension culture on the cell sediment to obtain an embryoid body; inoculating the embryoid bodies into a culture dish coated with Matrigel in advance, and inducing and differentiating in an inducing culture solution to obtain Neural Retina (NR) and RPE; and (3) picking up NR and RPE again, performing suspension culture to obtain 3D retina-like tissues including NR tissues, and then performing suspension culture in an optimized culture solution without retinoic acid, wherein NR differentiates all retinal cells including highly mature photoreceptor cells, Rhodopsin positive rod cells, L/M OPSIN positive red-green cone cells and S-OPSIN positive blue cone cells, and particularly obtains the retina-like tissues rich in red-green cones and rod cells.

Description

Method for obtaining retinal-like tissue rich in cones and rods by using human induced pluripotent stem cells

The technical field is as follows:

the invention belongs to the biological field of stem cell regeneration, and particularly relates to a method for regenerating a retina-like organ by using pluripotent stem cells to obtain retina seed cells.

Background

Retinal degenerative diseases are the major eye diseases that lead to irreversible blindness, mainly due to irreversible damage to the photoreceptor cells, RPE cells or ganglion cells. However, no effective treatment for this type of disease is currently available. The stem cell therapy brings new vision recovery hope for the diseases, obtains seed cells with similar characteristics to human retinal cells and sufficient quantity, and is the key of vision recovery treatment and disease mechanism research of retinal degenerative diseases.

Human retinal cells are mostly separated from aborted embryonic retinas in the past, but the number is very limited, the batch quality and the like are difficult to control, the requirements of clinical treatment cannot be met, and the development of retinal cell treatment technology is limited. In recent years, human retinal cells can also be directionally induced and differentiated from human pluripotent stem cells (hpscs) including Embryonic Stem Cells (ESCs) and induced pluripotent stem cells (ipscs), and a new source is provided for retinal seed cells. Currently, there are several differentiation-inducing protocols that direct the stepwise differentiation of hpscs towards retinal cells. Firstly, under the condition of classical adherence (namely two-dimensional, 2D) culture, hPSC not only differentiates a plurality of retinal cells including ganglion cells, RPE cells, photoreceptor cells and the like, but also can directly induce a primary vacuole-like structure which further differentiates a plurality of retinal cells; secondly, with the development of 3D suspension tissue induction culture systems, 3D retinal induction culture methods have emerged. Japanese scientists differentiated 3D vesicles from mouse and human ESCs, respectively, using the method of suspending EBs, which further differentiated various developing retinal cells; thirdly, the professor of Chinese scientist Zhongxiufeng adopts the induction culture condition of combining 2D and 3D to develop a set of retina induction differentiation system, firstly, hiPSCs are cultured in suspension to obtain embryoid EBs, then the EBs are differentiated in adherence again to obtain 3D retina tissue with a layered structure, including all main retina cells, and the photoreceptor cells with functions can be obtained after long-term culture.

Despite the great advances in the art of pluripotent stem cell-induced retina induction, there are some deficiencies in current retinal differentiation-inducing protocols. First, most induction protocols are complex, requiring the addition of various cytokines or signaling molecules, including retinoic acid a (RA); second, the retinal photoreceptor cells obtained are predominantly rod cells and few cone cells. Cones are responsible for intense or photopic, chromatic and fine vision, focusing on the macular area of the retina. Rod cells are responsible for night or scotopic vision, and are distributed around the periphery of the retina. Humans are primarily active during the day and, therefore, rely primarily on the function of the cones. Age-related macular degeneration has become the leading blinding eye disease in people over 50 years of age worldwide, mainly due to cone degeneration and death. Therefore, current retinal induction protocols are difficult to meet clinical therapeutic needs.

The invention content is as follows:

aiming at the problems of the existing retina induced differentiation technology (components are complex, and cone cells required by the treatment of retinal diseases are difficult to obtain), the invention provides an improved, simple and efficient induction scheme for obtaining the retina-like membrane rich in rods and cone cells.

The invention relates to a method for obtaining a retinal-like tissue rich in cones and rods by using human induced pluripotent stem cells, which comprises the steps of cloning and digesting hiPSCs to obtain cell precipitates, and performing suspension culture on the cell precipitates to obtain embryoid bodies; inoculating the embryoid body into a culture dish coated with Matrigel in advance, and inducing and differentiating in an inducing culture solution to obtain Neural Retinas (NR) and RPE; and (4) picking up the NR and the RPE again, and performing suspension culture to obtain the 3D retina including the NR tissue and the RPE. By using medium-and long-term suspension culture in the culture solution without RA, NR can develop all retinal cells, including highly mature photoreceptor cells, Rhodopsin positive rod cells, L/M OPSIN positive red-green cone cells and S-OPSIN positive blue cone cells, and particularly NR or retina-like tissues rich in rod and red-green cone cells are obtained.

The first object of the present invention is to provide a method for digesting clones of hiPSCs, wherein preferably, the step of digesting clones of hiPSCs to obtain cell pellet is a step of digesting clones of hiPSCs with EDTA to obtain cell pellet. Compared with dispase digestion, EDTA digestion has the following advantages: 1) the clone of the hiPSCs digested by EDTA can be gently blown and dispersed into small pieces, and the product digested by dispase is mostly small blocks, so that the size and the uniformity of the formation of EBs at the later stage are influenced; 2) simultaneously, digesting the same number of hiPSCs cells by using EDTA and dispase, wherein the EBs obtained after EDTA digestion is far greater than that obtained by dispase digestion; 3) EDTA is a chemical reagent and is a non-animal source, so that the EDTA is convenient for clinical transformation and application; dispase is protease, has difference among batches, is an animal source and is not beneficial to clinical transformation application; 4) the digestion time of EDTA is short, and the activity is stable and controllable at the digestion temperature of 37 ℃ for about 3-6 min; and dispase has long digestion time and unstable activity, needs 10-20min at the digestion temperature of 37 ℃, is not easy to master conditions and has poor repeatability.

Preferably, the digestion with EDTA is with 0.5. mu.M EDTA, at 37 ℃ for 5 min.

The second object of the present invention is to provide a simple and efficient method for induced differentiation of 3D retinas enriched in a large number of cone cells and rod cells from hiPSCs, which comprises obtaining 3D retinal tissue and then culturing the 3D retinal tissue in a culture medium, wherein the 3D retinal tissue is cultured in an optimized culture medium not containing retinoic acid. Mature photoreceptor cells develop in 3D retinal tissues after long-term culture, and about 43% of 3D retinal tissues are rich in L/M OPSIN positive red and green cone cells and are far higher than those reported in previous researches.

And (2) adding 10mM Blebbistatin into the digested hiPSCs cells for suspension culture to generate Embryoid Bodies (EBs), inoculating the embryoid bodies into a culture dish coated with Matrigel in advance, performing retinal induced differentiation by using a culture solution, picking up the differentiated retinas and RPE by using a Tungsten needle for suspension culture, and obtaining 3D retinal tissues which are clear in layers and rich in mature photoreceptor cells after long-term culture. The culture media used in sequence according to the induced differentiation process are mTeSR1, NIM culture solution, RDM culture solution, RC2 culture solution and RC1 culture solution, wherein mTeSR1 is hipSCs maintenance culture solution, NIM culture solution is early nerve induction culture solution, RDM culture solution is retina induction culture solution, and RC2 culture solution and RC1 culture solution are 3D retina tissue culture solution. The RC2 culture solution and the RC1 culture solution are RC2/RA culture solution and RC1/RA culture solution without retinoic acid. On one hand, the induction influence of exogenous molecules is reduced without adding retinoic acid, and on the other hand, the cost of the culture medium is saved (a large amount of culture solution is lost by adding RA to replace new culture solution every other day). In addition, retinoic acid is an important signaling molecule for the developmental differentiation of retinal photoreceptors, and previous studies have shown that high concentrations of RA inhibit the developmental maturation of cone cells. The culture medium without retinoic acid in the invention promotes the development and maturation of photoreceptor cells, particularly induces more red and green cone cells, and is expected to provide required seed cells for cell therapy of human macular degeneration.

The 3D retinal tissue is cultured in a culture solution without RA, namely the 3D retinal tissue is cultured in an RC2 culture solution and an RC1 culture solution in sequence, wherein the RC2 culture solution and the RC1 culture solution respectively refer to an RC2/RA culture solution without RA (retinoic acid) and an RC1/RA culture solution without RA.

The invention has the following beneficial effects:

the advantages of digesting hiPSCs with EDTA to initiate differentiation and prepare EBs are as follows: 1) the clone of the hiPSCs digested by EDTA can be gently blown and dispersed into small pieces, and the product digested by dispase is mostly small blocks, so that the size and the uniformity of the formation of EBs at the later stage are influenced; 2) simultaneously, digesting the same number of hiPSCs cells by using EDTA and dispase, wherein the EBs obtained after EDTA digestion is far greater than that obtained by dispase digestion; 3) EDTA is a chemical reagent and is a non-animal source, so that the EDTA is convenient for clinical transformation and application; dispase is protease, has difference among batches, is an animal source and is not beneficial to clinical transformation application; 4) the digestion time of EDTA is short, and the activity is stable and controllable at the digestion temperature of 37 ℃ for about 3-6 min; and dispase has long digestion time and unstable activity, needs 10-20min at the digestion temperature of 37 ℃, is not good in holding condition and has poor repeatability.

RA is a fat-soluble small molecule, is a metabolite of vitamin A (vitaminA), and has important significance in the process of embryonic development. RA plays an important role in the formation of visual primordia, cell proliferation and differentiation during ocular development. In addition, both in vivo and in vitro studies have found that RA has a significant regulatory role in the development of photoreceptor cells. Based on the above theory, in order to promote the development and maturation of photoreceptor cells, the induction scheme of differentiation from ESC or iPSCs to photoreceptors is adopted, and RA is more or less added to the culture medium as an inducer, so that the photoreceptor cells induced in the systems are mainly rod cells and few cone cells.

The 3D retinal differentiation system of the invention skillfully solves the problem, not only obtains the rod-like retinal tissue rich in the rod cells, but also obtains the retinal tissue rich in the red and green cone cells for the first time. Compared with other induced differentiation schemes, the novel scheme of the invention does not add small molecule inducer RA in the long-term culture process of the retinal tissue, and in the culture process of the system, the 3D retina still develops well, maintains a layered structure, develops main ganglion cells and intermediate neurons at the early stage, develops mature photoreceptor cells at the later stage, and has about half of 3D retinal tissue rich in red and green cone cells. Innovatively, RA does not need to add components in the process of in-vitro retinal photoreceptor cell development and maturation, and 3D retina develops tissues rich in red and green cone cells in the induction environment lacking RA. The advantages of the invention are 1) obtaining a large amount of retinal tissue rich in cone cells; 2) the addition of an exogenous component RA is reduced, and the clinical transformation application of an induction system is promoted; 3) the culture medium cost and the labor cost are saved, and the pollution risk is reduced. In previous studies, RA was extremely unstable, requiring frequent (and sometimes daily) replenishment and replacement of the culture medium, loss of large amounts of culture medium, requiring large amounts of manpower to maintain the cells, and increasing the risk of cell contamination.

Description of the drawings:

FIG. 1 is a graph comparing initial stages of digestion of HIPSCs with EDTA and dispase and total EB: panel A equivalent amounts of hipSCs were digested with EDTA (left panel) and dispase (right panel), respectively, to obtain similar amounts of cell pellet; panel B EB preparation was performed with EDTA (left panel) and dispase (right panel) digested hipSCs, respectively, and EB precipitation obtained with EDTA digested hipSCs after EB centrifugation on day six (left panel) was greater than that obtained with EDTA digested hipSCs (right panel).

FIG. 2 is a graph comparing the effect of EDTA and dispase digestion on EB formation: panel A, panel B and panel C are EB morphology and number of D0, D3 and D6, respectively, after digestion with EDTA; panel D, E and F show the EB morphology and number of D0, D3 and D6, respectively, after digestion with dispase, and the EB morphology obtained by EDTA digestion is relatively uniform and in large numbers.

Fig. 3 is that no RA optimized induction system does not affect 3D retinal early and intermediate differentiation: panels a and B show early expression of retinal precursor cell markers PAX6 and VSX2 in 3D retina (w6), panel C shows development of BRN3 positive ganglion cells in early 3D retina (w6), and panel D shows development of AP2 positive non-growing process cells in metaphase 3D retina (w 13).

FIG. 4 shows retinal-like tissues cultured for a long period without the RA-optimized induction culture system (34 weeks after differentiation). Panel A is a low power panel; FIG. B is an enlarged partial view of FIG. A showing the outer segment in the shape of a brush.

FIG. 5 is a graph showing immunofluorescence to show that a portion of retinal-like tissue cultured without the RA system is enriched with Rhodopsin-positive rod cells (w 21).

FIG. 6 is a graph of immunofluorescence showing that a portion of retinal-like tissue cultured without the RA system is rich in red/green cone cells: the immunohistochemistry results in panel A show that 3D retinal tissue is rich in a large number of L/M OPSIN-positive red-green cone cells, panel B is an enlarged view of the white square of panel A, panel C shows a reduction of Rhodopsin-positive rods in the same type of retinal tissue of panel A, and panel D is an enlarged view of the white square of panel C.

FIG. 7 shows immunofluorescence showing that cultured retinal-like tissues without the RA-optimized system contain a small number of S-OPSIN-positive blue cone cells (w 21).

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1:

maintenance culture of hiPSCs

1. hiPSCs cells: urine-derived human induced pluripotent stem cell, cell line UE017

2. Reagents and consumables:

1) mTeSR1 medium: STEM CELL, #05851, 4 ℃ C

2) EDTA: invitrogen 15575-038, normal temperature

3) dispase, sigma, D4693, Normal temperature

4) PBS (1X): jinuo biological medicine technology Co., Ltd, 14111202, Normal temperature

5)Matrigel：Corning，354277，-20℃

6) A six-hole plate: FALCON, 353046

7) Centrifuging the tube: BD FALCON, 352096

3. Instrument for measuring the position of a moving object

1)CO₂An incubator: SANYO, MCO-20A1C

2) And (3) inverting the microscope: nikon, TS100

4. The method comprises the following steps:

hiPSCs were maintained in mTeSR1 medium, passaged (about 4-5 days) at a density of up to about 80% -90% basal area, digested with 0.5mM EDTA or 1mg/mL dispase, and the digested cells plated on Matrigel-coated plates.

Second, the Effect of digestion patterns of hiPSCs on the formation of Embryoid Bodies (EBs)

1. Cell: step one cultured hiPSCs cells

2. Reagents and consumables:

1) mTeSR1 medium: STEM CELL, #05851, 4 ℃ C

2) NIM medium (formula shown in Table 1, and its preparation method comprises mixing the above components according to their content)

3) EDTA: 0.5M EDTA solution, Invitrogen, 15575-038, ambient temperature

4) dispase, sigma, D4693, Normal temperature

5)Blebbistatin：sigma

6)Matrigel：Corning，354277，-20℃

7) Low adsorption dish (100 mm):

3. the instrument comprises the following steps:

1)CO₂an incubator: SANYO, MCO-20A1C

4. The method comprises the following steps:

EB preparation by digestion of hiPSCs with EDTA: scraping differentiated cells in the hiPSCs clone obtained in the first step under an optical microscope, removing the residual culture solution by suction, washing the cells once by using sterile PBS, adding 0.5mM EDTA, incubating the cells for 5min at 37 ℃, gently blowing and beating the cells by using a gun head for 3-5 times, collecting cell suspension, centrifuging the cell suspension for 4min at 1000rpm, resuspending cell precipitates by using a culture solution containing 10 mu M of Blebbistatin, and performing suspension culture at 37 ℃ overnight.

Preparation of EBs from hipSCs by digestion with dispase: firstly scraping differentiated cells in the hiPSCs obtained in the first step under an optical microscope, removing the residual culture solution by suction, washing the cells once by using sterile PBS, adding 1mg/ml dispase, incubating the cells for 15min at 37 ℃, gently blowing and beating the cells by using a gun head for about 5 times, collecting cell suspension, centrifuging the cell suspension for 4min at 1000rpm, resuspending cell precipitates by using a culture solution containing 10 mu M of Blebbistatin, and carrying out suspension culture at 37 ℃ overnight.

Suspending the EDTA or dispase digested cell sediment in a culture solution containing 10 mu M of Blebbistatin respectively, transferring to a low adsorption dish, culturing at 37 ℃ overnight, marking the day of EBs preparation as day 0, marking the next day as day 1, and day 1, replacing the culture solution with a volume fraction of 25% NIM (the formula is shown in Table 1) + a volume fraction of 75% mTeSR 1; on day 2, the culture medium was replaced with NIM (50% by volume) and mTeSR1 (50% by volume), and all the cultures were replaced with NIM culture medium from the third day and were continuously cultured in suspension for 6-7 days. EBs formation was observed the next day, and suspension culture was continued for 6-7 days, centrifuged separately, and the pellet size was observed (FIG. 1).

As shown in FIG. 1, the same number of hipSCs were digested with EDTA or dispase, respectively, and FIG. 1-A shows that the cell pellet after digestion by both methods was substantially identical; FIG. 1-B shows that EB precipitated after 6 days of suspension, and EDTA digestion gave significantly higher amounts of EBs than dispse digestion.

FIG. 2 is a graph comparing the effect of EDTA and dispase digestion on EB formation, as shown in FIG. 2, the same number of hipSCs were digested with EDTA or dispase, FIG. 2-A and FIG. 2-D show the size and number of cell clumps on the day of EDTA and dispase digestion, respectively, the cells after EDTA digestion were more dispersed and uniform, while some of the products of dispase digestion were in the form of small pieces, and there was no significant difference in the number of cells observed on the day; FIGS. 2-B and 2-E are EBs after 3 days of aggregation, respectively, with significantly more EBs obtained by EDTA digestion than dispase; FIGS. 2-C and 2-F show EBs after 6 days, respectively, and the amount of EBs obtained by EDTA digestion was significantly greater than that of dispase.

Three, 3D retinal differentiation and RA-free formulation induced photoreceptor maturation

1. Cell: hiPSCs (UE017)

2. Reagents and consumables:

1) RDM culture solution: the formula is shown in table 2, and the preparation method comprises the steps of uniformly mixing the components according to the content;

2) RC2 culture solution: the formula is shown in table 3, and the preparation method comprises the steps of uniformly mixing the components according to the content;

3)RC1^-culture solution: the formula is shown in table 4, and the preparation method comprises the steps of uniformly mixing the components according to the content;

4) low adsorption 24-well plates or low adsorption dishes: BIOFIL, 170313-

3. The instrument comprises the following steps:

1)CO₂an incubator: SANYO, MCO-20A1C

4. The method comprises the following steps:

the hiPSCs were digested with the EDTA protocol described above and cultured in suspension to obtain EBs. The day of digestion was labeled day 0 (D0), the next day was labeled day 1, and so on. On day 6 or 7, EBs were re-seeded in petri dishes previously coated with Matrigel and adherent culture was initiated to induce differentiation. In the fourth differentiation period, NR and RPE regions were formed initially, and they were identified by inverted microscope, picked up by tungsten filament, transferred to 24-well low-adsorption plate or low-adsorption dish, and cultured in suspension for 2-3 times per week. Under the condition of suspension culture, NR grows thick and gradually forms a semitransparent ring, more or less RPE is attached to one end, the NR is called as 3D type retinal tissue, and can be cultured for a long time, and NR continues to differentiate, so that all retinal neuronal cells including photoreceptor cells are developmentally differentiated. The broth protocol was similar to the published article (reference Xiufeng Zhong, Nature Communications 2014), except that no RA was added, specifically: on days 6-15, neural induction was performed using NIM (table 1), on days 16-41 using RDM medium (table 2), from days 42-90, 3D retinas were cultured using RC2 medium with FBS (table 3), and from day 91 onwards using RC1 medium (table 4).

RA is generally considered to have an effect of regulating retinal development and photoreceptor cell maturation, and RA was added to the original protocol (protocol in the prior art, table 5RC2/RA culture solution and table 6RC1/RA culture solution, reference Xiufeng Zhong, Nature Communications 2014) for induction, and from day 42 to day 90 or day 63 to day 90, RC2 culture solution containing 1 μ M RA was used to change the solution every day or every other day (table 5, preparation method thereof is to mix the components uniformly according to their contents), and RC1 culture solution containing 0.5 μ M RA was changed every other day from more than 91 days (table 6, preparation method thereof is to mix the components uniformly according to their contents), so as to induce 3D retinal differentiation and photoreceptor maturation.

In the present invention, 3D retinal tissue was still selected to be cultured in RC2 and RC1 cultures at the two time points, respectively, but without the addition of RA, and the formulations of the specific cultures are shown in tables 3 and 4. 3-5 3D retinal tissues at the early stage are selected in each batch, fixed, dehydrated and sliced for immunofluorescence staining identification. The results indicate that 3D retinal development is unaffected in the RA-free induction system and follows the retinal developmental history as well. As can be seen in FIG. 3, in the protocol of the present invention, hipSCs also followed the developmental course of the retina, with early suspensory 3D retinas expressing early neuroretinal markers PAX6 and VSX2 (FIGS. 3-A and 3-B), mid-developmental formation of BRN 3-positive ganglion cells (FIG. 3-C), and AP 2-positive amacrine cells (FIG. 3-D).

After up to 17 weeks (up to 34 weeks) of culture, the 3D retinal tissue differentiated into mature photoreceptor cells. As can be seen in FIG. 4, FIG. 4-A is a 3D retina, approximately 34w retina, cultured for extended periods in RC2 and RC1 media; figure 4-B shows that the retinal tissue contains a large number of outer segment structures. 3-5 of 3D retinal tissues cultured for a long time are selected from each batch, fixed, dehydrated and sliced for immunofluorescence staining identification (general technology). As a result, it was found that about 50% of the 3D retinal tissue generated by the differentiation system of the present invention is dominated by Rhodopsin positive rods (7/14, n ═ 14), and fig. 5 shows that in the optimized culture environment without RA addition, 3D retina contains a large amount of Rhodopsin positive rods. More importantly, about 43% of the 3D retinal tissues contained a large number of L/M OPSIN positive red-green cone cells (6/14, n is 14), fig. 6-a is an environment without RA addition, the 3D retina contained a large number of L/M OPSIN positive cone cells, fig. 6-B is an enlarged view in the left white box, fig. 6-C shows a reduced proportion of Rhodopsin positive rod cells in the tissue, and fig. 6-D is an enlarged view in the left white box. FIG. 7 shows that in a culture environment without RA addition, the 3D retina contains a small number of S opsin-positive blue cone cells. More than 90% of the 3D retinal tissues obtained by the prior art system are Rhodopsin positive rod cells, a small amount of L/M OPSIN positive red-green cone cells and a small amount of S-OPSIN positive blue cone cells. Therefore, compared with the prior art, the system can obtain more retina tissues containing L/M OPSIN positive red and green cone cells, and the proportion of S-OPSIN positive blue cone cells is still smaller, which is basically consistent with the previous research.

TABLE 1NIM culture solution (day 3-15)

Ingredient	Amount(ml)	Final concentration
			DMEM/F12(1:1)(Gibco,C11330500BT)	500.00
100×N2(Gibco,17502-048)	5.00	1x
			Heparin(2mg/ml in PBS,)	0.50	2ug/ml
100×MEM-NEAA(Gibco,11140-050)	5.00	1x
				510.50

TABLE 2RDM culture solution (day 16-41)

TABLE 3RC2 culture solution (days 42-90) (inventive)

Ingredient	Amount(ml)	Final concentration
			DMEM/F12(1:1)(Gibco,C11330500BT)	250.00
DMEM basic(Gibco,C11995500BT)	175.00
			50×B27 without vitamin A(Gibco,12587-010)	10.00	1x
100×antibiotic and antimycotic(Gibco,15240)	5.00	1x
			100×MEM-NEAA(Gibco,11140-050)	5.00	1x
FBS(Gibco,10099-141)	50.00	10％
			100×GlutaMax(Gibco,35050-061)	5.00	1×
1000×Taurine(sigma,#T-0625)	0.50	100μM
				500.50

TABLE 4RC1 culture fluid (after 91 th day) (inventive)

Ingredient	Amount(ml)	Final concentration
			DMEM/F12-Glutamax(Gibco,C10565-018)	450.00
100×N2 supplement(Invitrogen,17502-048)	5.00	1x
			100×antibiotic and antimycotic(Gibco,15240)	5.00	1x
100×MEM-NEAA(Gibco,11140-050)	5.00	1x
			FBS(Gibco,10099-141)	50.00	10％
1000×Taurine(sigma,#T-0625)	0.50	100μM
				515.50

TABLE 5RC2/RA broth (days 42-90) (old protocol)

TABLE 6RC1/RA broth (after day 91) (old protocol)

Claims (4)

1. A method for obtaining a retinal-like tissue rich in cones and rods by using human induced pluripotent stem cells is characterized by comprising the steps of digesting a cell line UE017 to obtain a cell precipitate, suspending the cell precipitate in a Blebbistatin culture solution, marking the current day of preparation of an embryoid body as day 0, marking the next day as day 1, and replacing the culture solution with 25% NIM + 75% mTeSR1 by volume fraction on day 1; on day 2, the culture solution was replaced with NIM with a volume fraction of 50% and mTeSR1 with a volume fraction of 50%, all the culture solutions were replaced with NIM from the third day, suspension culture was continued for 6-7 days to obtain embryoid bodies, which were then inoculated into a petri dish previously coated with Matrigel, performing adherent induction culture with NIM culture solution on days 6-15, performing culture with RDM culture solution on days 16-41 to obtain 3D retina, wherein the 3D retina comprises NR tissue and RPE, performing culture with RC2 culture solution containing FBS on days 42-90, performing culture with RC1 culture solution after day 91, and allowing NR to develop into all retinal cells including highly mature photoreceptor cells, Rhodopsin positive rod cells, L/M OPSIN positive red-green cone cells and S-OPSIN positive blue cone cells, especially obtaining retinal tissue rich in red-green cone cells and rod cells;

the NIM culture solution comprises 500ml of 1:1 DMEM/F12, 5ml of 100 XN 2, 0.5ml of PBS solution containing 2mg/ml of Heparin and 5ml of 100 XMEM-NEAA;

the RDM culture solution comprises 300ml of 1:1 DMEM/F12, 200ml of DMEM basic, 10ml of 50 XB 27 with vitro vitamin A, 5ml of 100 XBtibiotic and antimycotic and 5ml of 100 XMEM-NEAA;

the RC2 culture solution comprises 250ml of 1:1 DMEM/F12, 175ml of DMEM basic, 10ml of 50 XB 27 with out vitamin A, 5ml of 100 × anti-biological and anti-biological, 5ml of 100 × MEM-NEAA, 50ml of FBS, 5ml of 100 × GlutaMax and 0.5ml of 1000 × Taurine;

the RC1 culture solution comprises 450ml of 1:1 DMEM/F12-Glutamax, 5ml of 100 xN 2 supplement, 5ml of 100 xAntibiotic and antimycotic, 5ml of 100 xMEM-NEAA, 50ml of FBS and 0.5ml of 1000 xTaurine.

2. The method of claim 1, wherein the digesting the cell line UE017 to obtain the cell pellet is digesting the cell line UE017 with EDTA to obtain the cell pellet.

3. The method of claim 2, wherein said EDTA digestion is with 0.5 μ M EDTA at 37 ℃ for 5 min.

4. A method for inducing and differentiating 3D retinas rich in a large number of cone cells and rod cells from hiPSCs with high efficiency and simplicity comprises obtaining 3D retinal tissue, and then culturing the 3D retinal tissue in a culture solution, wherein 3D retinas are cultured by RC2 culture solution added with FBS on days 1-48, and are cultured by RC1 culture solution after day 49;

the RC2 culture solution comprises 250ml of 1:1 DMEM/F12, 175ml of DMEM basic, 10ml of 50 XB 27 with out vitamin A, 5ml of 100 × anti-biological and anti-biological, 5ml of 100 × MEM-NEAA, 50ml of FBS, 5ml of 100 × GlutaMax and 0.5ml of 1000 × Taurine;

the RC1 culture solution comprises 450ml of 1:1 DMEM/F12-Glutamax, 5ml of 100 xN 2 supplement, 5ml of 100 xAntibiotic and antimycotic, 5ml of 100 xMEM-NEAA, 50ml of FBS and 0.5ml of 1000 xTaurine.

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