WO2023011251A1 - 快速高效的临床级色素上皮细胞诱导方法、试剂盒及应用 - Google Patents

快速高效的临床级色素上皮细胞诱导方法、试剂盒及应用 Download PDF

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WO2023011251A1
WO2023011251A1 PCT/CN2022/107890 CN2022107890W WO2023011251A1 WO 2023011251 A1 WO2023011251 A1 WO 2023011251A1 CN 2022107890 W CN2022107890 W CN 2022107890W WO 2023011251 A1 WO2023011251 A1 WO 2023011251A1
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medium
cells
inhibitor
cell culture
pathway inhibitor
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吴理达
顾雨春
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呈诺再生医学科技(珠海横琴新区)有限公司
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Definitions

  • the invention relates to the field of biotechnology, in particular to a fast and efficient clinical grade pigment epithelial cell induction method, kit and application.
  • RP retinal degenerative diseases
  • macular degeneration mainly include macular degeneration, and hereditary retinal degeneration.
  • RP retinitis pigmentosa
  • macular degeneration mainly includes macular degeneration, and hereditary retinal degeneration.
  • RPE retinal pigment epithelium
  • RP is a genetic disease, and more than 200 gene mutation sites related to RP have been found so far.
  • Macular degeneration is caused by genetic changes and environmental factors, and can be divided into juvenile macular degeneration and age-related macular degeneration (agerelated-macular degeneration, AMD) according to the age of onset.
  • RP and AMD are relatively common, and the incidence of RP is as high as 1/3000; while the incidence of AMD is more than 1/10 in people over 60 years old.
  • the drugs and methods used clinically to treat retinal degenerative diseases are very limited, most of which are anti-inflammatory therapy and nerve cell nutrition protective retinal degenerative disease drugs to delay the course of the disease, or drugs that inhibit angiogenesis to treat wet AMD, etc. .
  • the present invention provides a method for rapidly and efficiently inducing retinal pigment epithelial cells, through the three stages of ectoderm differentiation, pigment epithelial precursor cell differentiation, and retinal pigment epithelial cell maturation, directional induction of iPSCs greatly shortens the production time of RPE ; Each stage targets specific signaling pathways to promote iPSC differentiation.
  • the method provided by the invention can shorten the induction differentiation time, and can obtain RPE cells with relatively stable functions and vigor, which can finally be used for transplantation therapy.
  • the invention provides a medium for inducing retinal pigment epithelial cells, a medium combination, a kit and applications thereof.
  • the present invention provides a method for rapidly and efficiently inducing retinal pigment epithelial cells, the method comprising a step of inducing ectoderm differentiation and a step of inducing differentiation of pigment epithelial precursor cells.
  • the method further includes the step of inducing maturation of retinal pigment epithelial cells.
  • the method further includes the step of subculture of retinal pigment epithelial cells.
  • pigment epithelial cell In the present invention, “pigment epithelial cell”, “retinal pigment epithelial cell” and “RPE” all have the same meaning and can be used interchangeably.
  • the step of inducing ectoderm differentiation comprises using RDM1 medium and/or RDM2 medium to culture stem cells.
  • the stem cells include totipotent stem cells, pluripotent stem cells, and unipotent stem cells.
  • the stem cells are iPSC cells (induced pluripotent stem cells).
  • the iPSC cells can be commercialized cell lines, or can be induced from donor cells, including villi cells, skin (fibroblasts and keratinocytes), amniotic fluid, extraembryonic One or more of tissues (placenta and umbilical cord), umbilical cord blood, periosteum, dental tissue, adipose tissue, neural stem cells, liver cells, mesenchymal stem cells, peripheral blood cells, breast epithelial cells, adipose stem cells, umbilical cord stroma, and placenta.
  • donor cells including villi cells, skin (fibroblasts and keratinocytes), amniotic fluid, extraembryonic One or more of tissues (placenta and umbilical cord), umbilical cord blood, periosteum, dental tissue, adipose tissue, neural stem cells, liver cells, mesenchymal stem cells, peripheral blood cells, breast epithelial cells, adipose stem cells, umbilical cord strom
  • the donor may be human or non-human.
  • said non-human includes mammals (such as mice, monkeys, cows, sheep, pigs, horses, chickens).
  • mammals such as mice, monkeys, cows, sheep, pigs, horses, chickens.
  • the stem cells are human iPSC cells.
  • the basal medium of the RDM1 (“RDM1" and “RDM1 medium” can be used interchangeably to represent the same meaning in the present invention) is the RDM basal medium, and the RDM1 medium also includes the RDM basal medium other substances.
  • the basal medium of the RDM2 (“RDM2" and “RDM2 medium” can be used interchangeably to represent the same meaning in the present invention) is the RDM basal medium, and the RDM2 medium includes the RDM basal medium in addition to the RDM basal medium other substances.
  • the RDM basal medium is composed of DMEM/F12, KSR (KnockOut Serum Replacement, serum analog), Monothioglycerol Solution (human pluripotent stem cell serum-free medium), Chemically Defined Lipid Concentrate, at least a composition.
  • the RDM basal medium is composed of 88% DMEM/F12, 10% KSR, 5mM Monothioglycerol Solution, 1% Chemically Defined Lipid Concentrate, and 1% L-glutamine.
  • the DMEM/F12 can also be treated by one or more of William's E cell culture medium, Neurobasal Medium cell culture medium, MEM cell culture medium, DMEM cell culture medium, 1640RPMI cell culture medium, or F12 cell culture medium, etc. Hybrid substitution of species.
  • the KSR can also be replaced by other serum analogs.
  • said other serum analogs include but not limited to FBS (fetal bovine serum), horse serum, HAS (human serum albumin), BSA (bovine serum albumin).
  • the glutamine can be replaced by a glutamine substitute.
  • the glutamine substitute includes GlutaMAX TM Supplement.
  • DMEM/F-12 or “DMEM/F12” expresses the same meaning, which is a 1:1 mixture of DMEM and Ham's F-12; -12 broad components combined.
  • the DMEM/F12 includes a modified DMEM/F-12 medium prepared by adjusting components according to actual applications.
  • the DMEM/F-12 modified medium includes but is not limited to DMEM-low sugar-pyruvate-no glutamine-no phenol red, DMEM/F-12-GlutaMAX TM , DMEM/F-12 -HEPES (DMEM/F-12with HEPES), DMEM-low glucose-pyruvate-HEPES.
  • the DMEM/F-12 is a DMEM/F-12 with HEPES medium
  • the DMEM/F-12 with HEPES medium contains L-glutamine, HEPES, and phenol red.
  • the RDM1 medium also includes at least one of BMP signaling pathway inhibitors, Wnt pathway inhibitors, inhibitors of TGF- ⁇ type I receptors ALK5, ALK4 and ALK7, and ROCK pathway inhibitors .
  • the BMP signaling pathway inhibitor includes noggin, Dorsomorphin, DMH1, LDN-193189.
  • the BMP signaling pathway inhibitor includes 50-200 ng/mL noggin, 2-8 ⁇ M Dorsomorphin, 10-100 ⁇ M DMH1, 5 nM-5 ⁇ M LDN-193189.
  • the BMP signaling pathway inhibitor includes 50-200 ng/mL noggin, 2-8 ⁇ M Dorsomorphin, 10-100 ⁇ M DMH1, 5 nM-5 ⁇ M LDN-193189.
  • the BMP signaling pathway inhibitor includes 50 ng/mL noggin, 2 ⁇ M Dorsomorphin, and 3 ⁇ M LDN-193189.
  • the BMP signaling pathway inhibitor is selected from 50-200ng/mL noggin.
  • the BMP signaling pathway inhibitor is selected from 50ng/mL noggin.
  • the Wnt pathway inhibitor includes XAV-939, iCRT-3, iCRT-5, iCRT-14, IWP-4, IWR-1, wnt-C59.
  • the Wnt pathway inhibitor is 2-20 ⁇ M XAV-939.
  • the Wnt pathway inhibitor is 1 ⁇ M XAV-939.
  • the inhibitors of TGF- ⁇ type I receptors ALK5, ALK4 and ALK7 include LY2109761, A83-01, SB-525334, SD-208, EW-7197, Disitertide, LY3200882, SM16, SB431542.
  • the inhibitor of TGF- ⁇ type I receptors ALK5, ALK4 and ALK7 is 2-20 ⁇ M LY2109761.
  • the inhibitor of TGF- ⁇ type I receptors ALK5, ALK4 and ALK7 is 5 ⁇ M LY2109761.
  • the ROCK pathway inhibitor includes Thiazovivin and Y-27632.
  • the ROCK pathway inhibitor is 0.5-20 ⁇ M Thiazovivin.
  • the ROCK pathway inhibitor is Thiazovivin at 10 ⁇ M;
  • the RDM2 medium includes at least one of a WNT signaling pathway activator, a VEGFR kinase inhibitor, and a ROCK pathway inhibitor in addition to the RDM basal medium.
  • the WNT signaling pathway activator includes 6-bromoindirubin-3'-oxime (BIO).
  • the WNT signaling pathway activator is 1-20 ⁇ M 6-bromoindirubin-3'-oxime (BIO).
  • the WNT signaling pathway activator is 10 ⁇ M 6-bromoindirubin-3'-oxime.
  • the VEGFR kinase inhibitors include SU5402, AV-951, SU5205, SU5408.
  • the VEGFR kinase inhibitor is 1-20 ⁇ M SU5402.
  • the VEGFR kinase inhibitor is 2 ⁇ M SU5402.
  • the ROCK pathway inhibitor includes Thiazovivin and Y-27632.
  • the ROCK pathway inhibitor is 0.5-20 ⁇ M Thiazovivin.
  • the ROCK pathway inhibitor is 10 ⁇ M Thiazovivin.
  • the step of inducing the differentiation of pigment epithelial precursor cells comprises using RDM3 and/or RDM4 medium to cultivate RPE progenitor cells, that is, differentiated cells of ectoderm;
  • the RPE progenitor cells are cells cultured through the aforementioned method of inducing ectoderm differentiation.
  • pigment epithelial precursor cells are the precursor cells of the aforementioned “pigment epithelial cells”, “retinal pigment epithelial cells” and “RPE”.
  • the basal medium of the RDM3 (“RDM3" and “RDM3 medium” can be used interchangeably to represent the same meaning in the present invention) is the RDM basal medium, and the RDM3 medium also includes the RDM basal medium other substances.
  • the RDM3 medium further includes at least one of GSK signaling pathway inhibitors, VEGFR kinase inhibitors, ROCK pathway inhibitors, vitamins or vitamin analogs.
  • the GSK signaling pathway inhibitor includes 6-bromoindirubin-3'-oxime (BIO).
  • the GSK signaling pathway inhibitor is 1-20 ⁇ M 6-bromoindirubin-3'-oxime (BIO).
  • the GSK signaling pathway inhibitor is 10 ⁇ M 6-bromoindirubin-3'-oxime.
  • the VEGFR kinase inhibitors include SU5402, AV-951, SU5205, SU5408.
  • the VEGFR kinase inhibitor is 1-20 ⁇ M SU5402.
  • the VEGFR kinase inhibitor is 2 ⁇ M SU5402.
  • the ROCK pathway inhibitor includes Thiazovivin and Y-27632.
  • the ROCK pathway inhibitor is 0.5-20 ⁇ M Thiazovivin.
  • the ROCK pathway inhibitor is 10 ⁇ M Thiazovivin.
  • the vitamins or vitamin analogs include biotin, choline chloride, D-calcium pantothenate, folic acid, inositol, nicotinamide, pyridoxine hydrochloride, riboflavin, lime hydrochloride, coenzyme Q10 , putrescine dihydrochloride, vitamin A, vitamin B (Vitamin B), vitamin C, vitamin D, vitamin E, vitamin K, vitamin H, vitamin P, vitamin M, vitamin T, vitamin U, water-soluble vitamins.
  • the vitamins or vitamin analogs include Vitamin B.
  • Vitamin B3 More specifically, the vitamin or vitamin analog is Vitamin B3.
  • the vitamin or vitamin analog is 1-20mM Vitamin B3.
  • the vitamin or vitamin analog is 10 mM Vitamin B3.
  • the RDM4 medium is composed of DMEM/F12, KSR, N2 medium, glutamine and vitamins.
  • the DMEM/F12 or N2 medium can also be replaced by William's E cell culture medium, Neurobasal Medium cell culture medium, MEM cell culture medium, DMEM cell culture medium, 1640RPMI cell culture medium, or F12 cell culture medium, etc. A mixture of one or more alternatives.
  • the KSR can also be replaced by other serum analogs, including but not limited to FBS (fetal bovine serum), horse serum, HAS (human serum albumin), BSA (bovine serum albumin).
  • FBS fetal bovine serum
  • HAS human serum albumin
  • BSA bovine serum albumin
  • the glutamine can be replaced by a glutamine substitute.
  • the glutamine substitute includes GlutaMAX TM Supplement.
  • the RDM4 medium is composed of 89% DMEM/F12, 10% KSR, 1% N2 medium, 1% L-glutamine and 10mM Vitamin B3.
  • said inducing the maturation of retinal pigment epithelial cells comprises using RMM medium to culture pigment epithelial precursor cells.
  • the composition of the RMM medium includes DMEM/F12, B27 medium, and glutamine.
  • the RMM medium consists of 97% DMEM/F12, 2% B27 medium, and 1% L-glutamine.
  • the DMEM/F12 or B27 medium can also be replaced by William's E cell culture medium, Neurobasal Medium cell culture medium, MEM cell culture medium, DMEM cell culture medium, 1640RPMI cell culture medium, or F12 cell culture medium, etc. A mixture of one or more alternatives.
  • the KSR can also be replaced by other serum analogs, including but not limited to FBS (fetal bovine serum), horse serum, HAS (human serum albumin), BSA (bovine serum albumin).
  • FBS fetal bovine serum
  • HAS human serum albumin
  • BSA bovine serum albumin
  • the glutamine can be replaced by a glutamine substitute.
  • the glutamine substitute includes GlutaMAX TM Supplement.
  • the subculture of retinal pigment epithelial cells includes culturing mature retinal pigment epithelial cells using REM medium.
  • the composition of the REM medium includes DMEM/F12, KSR, glutamine, and ⁇ -mercaptoethanol.
  • the REM medium consists of 79% DMEM/F12, 20% KSR, 1% L-glutamine, and 50 ⁇ M ⁇ -mercaptoethanol.
  • the DMEM/F12 can also be treated by one or more of William's E cell culture medium, Neurobasal Medium cell culture medium, MEM cell culture medium, DMEM cell culture medium, 1640RPMI cell culture medium, or F12 cell culture medium, etc. Hybrid substitution of species.
  • the KSR can also be replaced by other serum analogs, including but not limited to FBS (fetal bovine serum), horse serum, HAS (human serum albumin), BSA (bovine serum albumin).
  • FBS fetal bovine serum
  • HAS human serum albumin
  • BSA bovine serum albumin
  • the glutamine can be replaced by a glutamine substitute.
  • the glutamine substitute includes GlutaMAX TM Supplement.
  • the ⁇ -mercaptoethanol can also be replaced by other reducing agents, including but not limited to ⁇ -mercaptoethanol, dithiothreitol, dithioerythritol, reduced glutathione, Cysteine, thiocarbamate, sodium dithiosulfinate, ascorbate, tin dichloride, or sodium borohydride.
  • reducing agents including but not limited to ⁇ -mercaptoethanol, dithiothreitol, dithioerythritol, reduced glutathione, Cysteine, thiocarbamate, sodium dithiosulfinate, ascorbate, tin dichloride, or sodium borohydride.
  • the concentrations marked in the present invention are all final concentrations, and all percentages are volume percentages.
  • the frequency of changing the liquid when using any medium of RDM1, RDM2, RDM3, RDM4, RMM or REM is adjusted according to the growth state of the cells; preferably, the frequency of changing the liquid is daily.
  • the total number of days for using the RDM1 medium is 3-9 days.
  • the total number of days for using the RDM1 medium is 6 days.
  • the total number of days for using the RDM2 medium is 2-8 days.
  • the total number of days for using the RDM2 medium is 5 days.
  • the total number of days for using the RDM3 medium is 1-7 days.
  • the total number of days for using the RDM3 medium is 4 days.
  • the total number of days for using the RDM4 medium is 3-9 days.
  • the total number of days for using the RDM4 medium is 6 days.
  • the total number of days for using the RMM medium is 6-12 days.
  • the total number of days for using the RMM medium is 9 days.
  • the method further includes the step of cell detection.
  • the cell detection can be one of cell viability detection, immune-based detection, flow cytometry detection, colorimetric detection, detection based on gold nanoparticles, fluorescence detection, ultraviolet detection, and detection of cell markers or more.
  • the method uses a cell culture method commonly used by those skilled in the art to process cells, and the cell culture is cell preparation in vitro using culture medium, cell sorting, cell clone culture, cell expansion culture, cell enrichment , cell purification, cell engineering, three-dimensional cell culture, cell fermentation, tissue culture, organ culture in any form.
  • the cell culture can be carried out in an incubator, or in other environments suitable for cell growth.
  • the incubator is a CO2 incubator.
  • the incubator is a constant temperature incubator; more preferably, the constant temperature is 37°C.
  • the present invention provides a method for rapidly and efficiently inducing RPE progenitor cells, the method comprising culturing stem cells in a medium containing a small molecule compound.
  • the small molecule compounds include BMP signaling pathway inhibitors, Wnt pathway inhibitors, inhibitors of TGF- ⁇ type I receptors ALK5, ALK4 and ALK7, ROCK pathway inhibitors, WNT signaling pathway activators, any of the VEGFR kinase inhibitors one or more.
  • the medium containing the small molecule compound is the aforementioned RDM1 medium and/or RDM2 medium.
  • the present invention provides a method for inducing pigment epithelial precursor cells, the method comprising using a medium containing a small molecule compound for cell culture.
  • the small molecular compound includes any one or more of GSK signaling pathway inhibitors, VEGFR kinase inhibitors, ROCK pathway inhibitors, vitamins or vitamin analogs.
  • the medium containing the small molecule compound is the RDM3 medium according to claim 3.
  • said cell culture is the culture of RPE progenitor cells.
  • the RPE progenitor cells are prepared by the aforementioned method for rapidly and efficiently inducing RPE progenitor cells.
  • the method further comprises culturing the cells using the aforementioned RDM4 medium.
  • the present invention provides a culture medium selected from any one of the following: the aforementioned RDM1 medium, RDM2 medium, RDM3 medium, RDM4 medium, RMM medium, and REM medium.
  • the present invention provides a combination of culture media, which is selected from any combination of the following: RDM1 medium, RDM2 medium, RDM3 medium, RDM4 medium, RMM medium, REM medium Medium.
  • the present invention provides a kit for inducing retinal pigment epithelial cells, said kit comprising at least one of the following substances: BMP signaling pathway inhibitors, Wnt pathway inhibitors, TGF- ⁇ type I receptors ALK5, ALK4 and ALK7 Inhibitors of ROCK pathway inhibitors, WNT signaling pathway activators, VEGFR kinase inhibitors, GSK signaling pathway inhibitors, VEGFR kinase inhibitors, vitamins and their analogs.
  • the kit includes reagents for preparing any one or more of the aforementioned RDM1 medium, RDM2 medium, RDM3 medium, RDM4 medium, RMM medium, and REM medium.
  • the RDM1 medium, RDM2 medium, RDM3 medium, RDM4 medium, RMM medium, and REM medium described in the present invention can be artificially prepared medium or commercialized medium.
  • the kit also includes instruments required for culturing cells.
  • the instruments include but are not limited to culture vessels (such as culture plates, culture dishes, culture bottles), incubators (including CO2 incubators), biological safety cabinets, centrifuges, water bath instruments, refrigerators, pure water equipment, Microscopes, drying cabinets, cell cryopreservators, sterilizers.
  • culture vessels such as culture plates, culture dishes, culture bottles
  • incubators including CO2 incubators
  • biological safety cabinets including centrifuges, water bath instruments, refrigerators, pure water equipment, Microscopes, drying cabinets, cell cryopreservators, sterilizers.
  • the present invention provides the aforementioned RDM1 medium, RDM2 medium, RDM3 medium, RDM4 medium, RMM medium, REM medium, medium combination, kit, BMP signaling pathway inhibitor, Wnt pathway inhibitor , inhibitors of TGF- ⁇ type I receptors ALK5, ALK4 and ALK7, ROCK pathway inhibitors, WNT signaling pathway activators, VEGFR kinase inhibitors, GSK signaling pathway inhibitors, any one of vitamins and their analogs in the induction Applications in the retinal pigment epithelium.
  • the present invention provides the retinal pigment epithelial cells prepared by the aforementioned method and their application in the preparation of ophthalmic disease medicines.
  • the present invention provides a method for treating ophthalmic diseases, the method for treating ophthalmic diseases comprises preparing retinal pigment epithelial cells by the aforementioned method.
  • the method for treating ophthalmic diseases further includes cell transplantation.
  • the ophthalmic diseases include retinal degenerative diseases; the main symptoms of the retinal degenerative diseases include irreversible loss of retinal pigment epithelial cells, and eventually lead to loss of visual function.
  • the retinal degenerative diseases mainly include retinitis pigmentosa (retinitis pigmentosa, RP), macular degeneration, Leber's disease (also known as Leber congenital amaurosis, Leber congenital amaurosis), Usher syndrome, retinal atrophy (including lesion damage Retinal flinching caused by retinal or genetic factors).
  • retinitis pigmentosa retinitis pigmentosa, RP
  • macular degeneration retinitis pigmentosa
  • Leber's disease also known as Leber congenital amaurosis, Leber congenital amaurosis
  • Usher syndrome retinal atrophy (including lesion damage Retinal flinching caused by retinal or genetic factors).
  • the macular degeneration includes juvenile macular degeneration (Stargarde, also called congenital macular degeneration) and age-related macular degeneration (age related-macular degeneration, AMD).
  • juvenile macular degeneration also called congenital macular degeneration
  • age-related macular degeneration age related-macular degeneration, AMD
  • the present invention provides the following applications:
  • BMP signaling pathway inhibitors BMP signaling pathway inhibitors, Wnt pathway inhibitors, TGF- ⁇ type I receptor ALK5, ALK4 and ALK7 inhibitors, ROCK pathway inhibitors, WNT signaling pathway activators, VEGFR kinase inhibitors, GSK signaling pathway inhibitors, Application of any one of vitamins and their analogs in inducing retinal pigment epithelial cells;
  • BMP signaling pathway inhibitors BMP signaling pathway inhibitors, Wnt pathway inhibitors, TGF- ⁇ type I receptor ALK5, ALK4 and ALK7 inhibitors, ROCK pathway inhibitors, WNT signaling pathway activators, VEGFR kinase inhibitors, GSK signaling pathway inhibitors, Application of any one of vitamins and their analogs in inducing retinal pigment epithelial cells;
  • RDM1 medium RDM2 medium, BMP signaling pathway inhibitors, Wnt pathway inhibitors, TGF- ⁇ type I receptor ALK5, ALK4 and ALK7 inhibitors, ROCK pathway inhibitors, WNT signaling pathway activators, VEGFR kinase inhibitors agent, the application of the aforementioned kit in inducing RPE progenitor cells;
  • RDM3 medium Application of RDM3 medium, RDM4 medium, GSK signaling pathway inhibitor, VEGFR kinase inhibitor, ROCK pathway inhibitor, vitamins or vitamin analogs, and the aforementioned kits in inducing pigment epithelial precursor cells.
  • the present invention provides the following cell populations:
  • a cell population prepared by the aforementioned method for inducing RPE progenitor cells the proportion of cells expressing PAX6 and RPE65 in the cell population is at least 5%; preferably, at least 10%;
  • the cell population prepared by the aforementioned method of inducing pigment epithelial precursor cells is at least 10%; preferably, at least 20%;
  • Figure 1 shows the changes in cell morphology under a 4x optical microscope on day 2, day 7 and day 12; A: day 2, B: day 7, C: day 14.
  • Figure 2 is the morphological changes of cells under a 4x optical microscope on the 14th day, the 18th day and the 24th day; A: the 14th day, B: the 18th day, C: the 24th day.
  • Figure 3 shows the cell morphology changes under the light microscope at 4X, 10X and 20X on day 52; A: 4X, B: 10X, C: 20X.
  • Figure 4 is the overall morphology of the cells on day 52 under the naked eye.
  • Figure 5 is the detection of PAX6 and RPE65 double positive cells on day 6, day 12 and day 24; the first column is DAPI staining, the second column is PAX6 staining, the third column is RPE65 staining, and the fourth column is PAX6 and RPE65 fusion.
  • Fig. 6 is a graph showing the statistical results of relative mRNA expression of RPE progenitor cell-related genes on the 12th day.
  • Figure 7 is the result of immunofluorescence detection of RPE cell-related genes on day 36; A is the cell morphology under white light, B is the result of ZO1 staining, C is the result of PAX6 and RPE65 staining, D is the result of ZO1 and CRALBP staining picture.
  • Fig. 8 is a graph showing the statistical results of relative mRNA expression of RPE cell-related genes on day 36.
  • Fig. 9 is a graph showing the statistical results of the relative expression of PAX6 mRNA in cells on day 6 when different BMP inhibitors were used.
  • Counterstain nuclei add DAPI dropwise and incubate in the dark for 5 minutes, stain the specimens for nuclei, wash off excess DAPI with PBST 5min ⁇ 4 times;
  • Embodiment 1 RPE differentiation process and test results
  • iPSC cells On day 0, seed iPSC cells at 1.0 ⁇ 10 3 -5.0 ⁇ 10 5 /cm 2 , in this case, seed cells at a density of 5.0 ⁇ 10 3 cells/cm 2 , and culture the cells at 37°C/5% In a CO 2 cell incubator, the medium used is RDM1.
  • the basal medium of RDM1 consists of 88% DMEM/F12, 10% KSR, 5mM Monothioglycerol Solution, 1% Chemically Defined Lipid Concentrate, 1% L-Glutamine.
  • the RDM1 medium also contains:
  • BMP signaling pathway inhibitor 50ng/mL noggin
  • the ROCK pathway inhibitor is 10uM Thiazovivin.
  • the basal medium of RDM2 consists of 88% DMEM/F12, 10% KSR, 5mM Monothioglycerol Solution, 1% Chemically Defined Lipid Concentrate, 1% L-Glutamine composition.
  • the RDM2 medium also contains:
  • WNT signaling pathway activator 10 ⁇ M 6-bromoindirubin-3′-oxime (BIO).
  • VEGFR kinase inhibitor 2 ⁇ M SU5402
  • ROCK pathway inhibitor Thiazovivin at 10 ⁇ M.
  • successful differentiation is marked by the generation of at least 10% PAX6 and RPE65 double-positive cells.
  • the detection method see General Method 1.
  • the detection results are shown in the second row of Figure 5; or the expression levels of PAX6, RPE65, IHX2, and pmel17 are increased by 5 times.
  • general method 2 for the detection method see general method 2 for the detection method, and the results are shown in Figure 6.
  • the basal medium of RDM3 consists of 88% DMEM/F12, 10% KSR, 5mM Monothioglycerol Solution, 1% Chemically Defined Lipid Concentrate, 1% L-Glutamine composition.
  • the RDM3 medium also contains:
  • GSK signaling pathway inhibitor 10 ⁇ M 6-bromoindirubin-3'-oxime (BIO);
  • VEGFR kinase inhibitor 2 ⁇ M SU5402;
  • ROCK pathway inhibitor Thiazovivin at 10 ⁇ M
  • Vitamins and their analogues 10 mM Vitamin B3.
  • the basal medium of RDM4 is composed of 89% DMEM/F12, 10% KSR, 1% N2 medium, 1% L-glutamine and 10mM Vitamin B3 .
  • successful differentiation is marked by the generation of at least 20% of PAX6 and RPE65 positive cells.
  • For the detection method see General Method 1. The detection results are shown in the third row of FIG. 5 .
  • the basal medium of RMM is composed of 97% DMEM/F12, 2% B27 medium, and 1% L-glutamine.
  • successful differentiation is marked by the production of at least 80% positive cells for ZO-1, RPE65, Pax6, and CRALBP, and no less than 1% of the cells produce melanin. See general method 1 for the detection method, and the test results are shown in Figure 7; or qPCR detects that the expression levels of TYRP2, PEDF, PMEL17, RPE65, and CRALBP are increased by at least 100 times, and the detection method is shown in General method 2, and the test results are shown in Figure 8.
  • the REM medium is composed of 79% DMEM/F12, 20% KSR, 1% L-glutamine, and 50 ⁇ M ⁇ -mercaptoethanol.
  • the cell morphology changes were observed under a 4x optical microscope.
  • the cell morphology on the 2nd day, 7th day and 12th day is shown in Figure 1.
  • the cells showed colony growth, on the seventh day the cells expanded and showed obvious epithelioid morphology, and on the 12th day some epithelioid cells began to accumulate and grow.
  • the morphology of the cells on the 14th day, the 18th day and the 24th day is shown in Fig. 2 .
  • the epithelioid cells showed colony accumulation growth
  • the epithelioid cells showed colony accumulation growth, the cell shape changed, and the cell boundary became more distinct.
  • the epithelioid cells showed colony accumulation growth, and the cell shape showed irregular polygons.
  • the epithelioid cells showed colony accumulation and growth, the cell shape changed, and the cell boundaries became more distinct.
  • the 20X optical microscope the cells showed a typical regular hexagonal shape.
  • the overall morphology of the cells under the naked eye is shown in Figure 4. About 80% of the cells showed black pigmentation under the naked eye.
  • Figure 5 shows the detection of PAX6 and RPE65 double positive cells on day 6, day 12 and day 24.
  • successful differentiation produces at least 5% of PAX6 and RPE65 double positive cells; on day 12, successful differentiation produces at least 10% of PAX6 and RPE65 double positive cells; on day 24, successful differentiation produces at least 20% of PAX6 and RPE65 double positive cells cell.
  • Figure 6 is the detection of genes related to RPE progenitor cells on the 12th day.
  • the expression levels of PAX6, RPE65, IHX2, and pmel17 were at least 5 times higher than those of iPSCs.
  • Both OCT4 and NANOG are iPSC marker genes, with lower expression in RPE progenitor cells.
  • Figure 7 is the detection of RPE cell-related genes on the 36th day. Differentiate to produce at least 80% positive cells for ZO-1, RPE65, Pax6, CRALBP
  • Figure 8 is the detection of RPE cell-related genes on the 36th day.
  • the expression levels of TYRP2, PEDF, PMEL17, RPE65, and CRALBP were at least 100-fold higher than that of iPSCs.
  • the iPSC cells were cultured according to the method of Example 1, only 50ng/mL noggin or 2 ⁇ M Dorsomorphin or 3 ⁇ M LDN-193189 were added to the RDM1 medium as three parallel controls, and the PAX6 expression level of the cells was detected on the 6th day .

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Abstract

一种快速高效的临床级色素上皮细胞诱导方法、试剂盒及应用。提供了一种快速高效诱导视网膜色素上皮细胞的方法，通过3个阶段，定向诱导iPSC，极大的缩短了RPE产生时间；具体地，所述方法包括使用含有小分子化合物的培养基进行细胞培养，所述小分子化合物包括BMP信号通路抑制剂，Wnt通路抑制剂，TGF-βI型受体ALK5、ALK4和ALK7的抑制剂，ROCK通路抑制剂，WNT信号通路激活剂，VEGFR激酶抑制剂，GSK信号通路抑制剂，VEGFR激酶抑制剂，维生素及其类似物。

Description

快速高效的临床级色素上皮细胞诱导方法、试剂盒及应用 技术领域
本发明涉及生物技术领域，具体涉及快速高效的临床级色素上皮细胞诱导方法、试剂盒及应用。
背景技术
眼是人类的重要器官，而视网膜退行性疾病可致盲，给患者带来极大的痛苦。视网膜退行性疾病主要包括视网膜色素变性(retinitis pigmentosa，RP)、黄斑变性以及遗传性视网膜退行等，这些疾病存在着不同的症状以及相关致病基因或易感基因。尽管这些疾病的病因和病理进程各不相同，但都存在视网膜细胞数量的进行性减少，主要包括视网膜色素上皮细胞(retinal pigment epithelium，RPE)和光感受器细胞的不可逆损失，并最终导致视觉功能丧失。RP为遗传性疾病，目前已发现200多个与RP相关的基因突变位点。黄斑变性是由基因改变和环境因素共同引起，根据患病年龄分为少年黄斑变性和年龄相关性黄斑变性(agerelated-macular degeneration，AMD)。临床上RP和AMD较为普遍，其中RP的发病率高达1/3000；而AMD在大于60岁的人群中的发病率超过1/10。目前，临床上用来治疗视网膜退行性疾病的药物和方法非常有限，多数为抗炎治疗及神经细胞营养保护性视网膜退行性疾病药物用以延缓病程，或者抑制血管生长类药物以治疗湿性AMD等。
但是药物治疗无法恢复已经受损的视神经细胞和功能RPE细胞，而利用细胞移植手段则可以有针对性地将有特定功能的细胞移植并整合到视网膜中以恢复其受损功能，应用前景更为广泛。目前，细胞移植是治疗退行性眼病的重要方法之一。针对视网膜退行性疾病的细胞替代疗法形式的再生医学具有广阔的前景，因为无论潜在的遗传原因或后天原因，均可使用相同的治疗剂。现代干细胞技术已经产生了临床级别的细胞疗法，目前正在研究人类胚胎干细胞(hESCs)和诱导性多能干细胞(iPSCs)治疗视网膜变性。目前的分化方法存在的主要不足在于诱导分化耗时长(90-140天)，以及移植后RPE(视网膜色素上皮细胞)的功能性差和存活率低。
发明内容
本发明提供了一种快速高效诱导视网膜色素上皮细胞的方法，通过外胚层分化、色素上皮前体细胞分化、视网膜色素上皮细胞成熟这3个阶段，定向诱导iPSC，极大的缩短了RPE产生时间；每个阶段针对特定的信号通路，促进iPSC分化。
本发明所提供的方法能够缩短诱导分化时间，并且能得到功能和活力均比较稳定的RPE细胞，最终可以用于移植治疗。同时，本发明提供了诱导视网膜色素上皮细胞的培养基、培养基组合、试剂盒及其应用。
第一方面，本发明提供了一种快速高效诱导视网膜色素上皮细胞的方法，所述方法包括诱导外胚层分化的步骤和诱导色素上皮前体细胞分化的步骤。
优选地，所述方法还包括诱导视网膜色素上皮细胞成熟的步骤。
优选地，所述方法还包括视网膜色素上皮细胞传代的步骤。
本发明中“色素上皮细胞”“视网膜色素上皮细胞”“RPE”均表示相同含义，可以互换使用。
在一种实施方式中，所述诱导外胚层分化的步骤包括使用RDM1培养基和/或RDM2培养基培养干细胞。
优选地，所述干细胞包括全能干细胞、多能干细胞、单能干细胞。
优选地，所述干细胞是iPSC细胞(诱导性多能干细胞)。
优选地，所述iPSC细胞可以是商品化的细胞系，也可以是由供体细胞诱导而来，所述供体细胞包括绒毛细胞、皮肤(成纤维细胞和角质形成细胞)、羊水、胚外组织(胎盘和脐带)、脐带血、骨膜、牙组织、脂肪组织、神经干细胞、肝细胞、间质干细胞、外周血细胞、乳腺上皮细胞、脂肪干细胞、脐带基质和胎盘中的一种或多种。
优选地，所述供体可以是人或非人。
优选地，所述非人包括哺乳动物(如鼠、猴、牛、羊、猪、马、鸡)。
优选地，所述干细胞是人源的iPSC细胞。
优选地，所述RDM1(本发明中“RDM1”和“RDM1培养基”可以互换使用，表示相同含义)的基础培养基是RDM基础培养基，所述RDM1培养基除RDM基础培养基还包括其他物质。
优选地，所述RDM2(本发明中“RDM2”和“RDM2培养基”可以互换使用，表示相同含义)的基础培养基是RDM基础培养基，所述RDM2培养基除RDM基础培养基还包括其他物质。
优选地，所述RDM基础培养基是由DMEM/F12，KSR(KnockOut Serum Replacement，血清类似物)，Monothioglycerol Solution(人多能性干细胞无血清培养基)，Chemically Defined Lipid Concentrate，谷氨酰胺中至少一种组成。
更优选地，所述RDM基础培养基是由88％DMEM/F12，10％KSR，5mM Monothioglycerol Solution，1％Chemically Defined Lipid Concentrate，1％L-谷氨酰胺组成。
优选地，所述DMEM/F12还可以被William’s E细胞培养基、Neurobasal Medium细胞培养基、MEM细胞培养基、DMEM细胞培养基、1640RPMI细胞培养基，或F12细胞培养基等中的一种或多种的混合替代。
优选地，所述KSR还可以被其他血清类似物替代。
优选地，所述其他血清类似物包括但不限于FBS(胎牛血清)、马血清、HAS(人血清白蛋白)、BSA(牛血清蛋白)。
优选地，所述谷氨酰胺可以更换为谷氨酰胺代替物。
优选地，所述谷氨酰胺代替物包括GlutaMAX TM Supplement。
如本发明所述“DMEM/F-12”或“DMEM/F12”表达相同含义，其是一种1:1DMEM和Ham's F-12混合物；该配方将DMEM的高浓度葡萄糖、氨基酸及维生素与F-12的广泛组分结合在一起。
优选地，所述DMEM/F12包括根据实际应用对成分进行调整而制成的DMEM/F-12改良培养基。
优选地，所述DMEM/F-12改良培养基包括但不限于DMEM-低糖-丙酮酸盐-不含谷氨酰胺-不含酚红、DMEM/F-12-GlutaMAX TM、DMEM/F-12-HEPES(DMEM/F-12with HEPES)、DMEM-low glucose-pyruvate-HEPES。
优选地，所述DMEM/F-12是DMEM/F-12 with HEPES培养基，所述DMEM/F-12 with HEPES培养基中包含L-谷氨酰胺、HEPES、酚红。
优选地，所述RDM1培养基除RDM基础培养基还包括BMP信号通路抑制剂，Wnt通路抑制剂，TGF-βI型受体ALK5、ALK4和ALK7的抑制剂，ROCK通路抑制剂中的至少一种。
优选地，所述BMP信号通路抑制剂包括noggin、Dorsomorphin、DMH1、LDN-193189。
优选地，所述BMP信号通路抑制剂包括50-200ng/mL的noggin、2-8μM的Dorsomorphin、10-100μM的DMH1、5nM-5μM的LDN-193189。
优选地，所述BMP信号通路抑制剂包括50-200ng/mL的noggin、2-8μM的Dorsomorphin、10-100μM的DMH1、5nM-5μM的LDN-193189。
优选地，所述BMP信号通路抑制剂包括50ng/mL的noggin、2μM的Dorsomorphin、3μM的LDN-193189。
优选地，所述BMP信号通路抑制剂选用50-200ng/mL的noggin。
优选地，所述BMP信号通路抑制剂选用50ng/mL的noggin。
优选地，所述Wnt通路抑制剂包括XAV-939、iCRT-3、iCRT-5、iCRT-14、IWP-4、IWR-1、wnt-C59。
优选地，所述Wnt通路抑制剂为2-20μM的XAV-939。
优选地，所述Wnt通路抑制剂为1μM的XAV-939。
优选地，所述TGF-βI型受体ALK5、ALK4和ALK7的抑制剂包括LY2109761、A83-01、SB-525334、SD-208、EW-7197、Disitertide、LY3200882、SM16、SB431542。
优选地，所述TGF-βI型受体ALK5、ALK4和ALK7的抑制剂是2-20μM的LY2109761。
优选地，所述TGF-βI型受体ALK5、ALK4和ALK7的抑制剂是5μM的LY2109761。
优选地，所述ROCK通路抑制剂包括Thiazovivin、Y-27632。
优选地，所述ROCK通路抑制剂是0.5-20μM的Thiazovivin。
优选地，所述ROCK通路抑制剂是10μM的Thiazovivin；
优选地，所述RDM2培养基除RDM基础培养基还包括WNT信号通路激活剂，VEGFR激酶抑制剂、ROCK通路抑制剂中的至少一种。
优选地，所述WNT信号通路激活剂包括6-bromoindirubin-3′-oxime(BIO)。
优选地，所述WNT信号通路激活剂为1-20μM的6-bromoindirubin-3′-oxime(BIO)。
优选地，所述WNT信号通路激活剂为10μM的6-bromoindirubin-3′-oxime。
优选地，所述VEGFR激酶抑制剂包括SU5402、AV-951、SU5205、SU5408。
优选地，所述VEGFR激酶抑制剂为1-20μM的SU5402。
优选地，所述VEGFR激酶抑制剂为2μM的SU5402。
优选地，所述ROCK通路抑制剂包括Thiazovivin、Y-27632。
优选地，所述ROCK通路抑制剂为0.5-20μM的Thiazovivin。
优选地，所述ROCK通路抑制剂为10μM的Thiazovivin。
在一种实施方式中，所述诱导色素上皮前体细胞分化的步骤包括使用RDM3和/或RDM4培养基培养RPE祖细胞，也就是外胚层分化了的细胞；
优选地，所述RPE祖细胞是经过了前述诱导外胚层分化的方法培养的细胞。
本发明中所述的“色素上皮前体细胞”也就是前述“色素上皮细胞”“视网膜色素上皮细胞”“RPE”的前体细胞。
优选地，所述RDM3(本发明中“RDM3”和“RDM3培养基”可以互换使用，表示相同含义)的基础培养基是RDM基础培养基，所述RDM3培养基除RDM基础培养基还包括其他物质。
优选地，所述RDM3培养基除RDM基础培养基还包括GSK信号通路抑制剂，VEGFR激酶抑制剂，ROCK通路抑制剂，维生素或者维生素类似物中的至少一种。
优选地，所述GSK信号通路抑制剂包括6-bromoindirubin-3′-oxime(BIO)。
优选地，所述GSK信号通路抑制剂为1-20μM的6-bromoindirubin-3′-oxime(BIO)。
优选地，所述GSK信号通路抑制剂为10μM的6-bromoindirubin-3′-oxime。
优选地，所述VEGFR激酶抑制剂包括SU5402、AV-951、SU5205、SU5408。
优选地，所述VEGFR激酶抑制剂为1-20μM的SU5402。
优选地，所述VEGFR激酶抑制剂为2μM的SU5402。
优选地，所述ROCK通路抑制剂包括Thiazovivin、Y-27632。
优选地，所述ROCK通路抑制剂为0.5-20μM的Thiazovivin。
优选地，所述ROCK通路抑制剂为10μM的Thiazovivin。
优选地，所述维生素或者维生素类似物包括生物素、氯化胆碱、D-泛酸钙、叶酸、肌醇、烟酰胺、吡哆醇盐酸盐、核黄素、盐酸石克氨、辅酶Q10、腐胺二盐酸盐、维生素A、维生素B(Vitamin B)、维生素C、维生素D、维生素E、维生素K、维生素H、维生素P、维生素M、维生素T、维生素U、水溶性维生素。
优选地，所述维生素或者维生素类似物包括Vitamin B。
更具体地，所述维生素或者维生素类似物是Vitamin B3。
优选地，所述维生素或者维生素类似物是1-20mM的Vitamin B3。
优选地，所述维生素或者维生素类似物是10mM的Vitamin B3。
优选地，所述RDM4培养基是DMEM/F12，KSR，N2培养基，谷氨酰胺以及维生素组成。
优选地，所述DMEM/F12或N2培养基还可以被William’s E细胞培养基、Neurobasal Medium细胞培养基、MEM细胞培养基、DMEM细胞培养基、1640RPMI细胞培养基，或F12细胞培养基等中的一种或多种的混合替代。
优选地，所述KSR还可以被其他血清类似物替代，所述其他血清类似物包括但不限于FBS(胎牛血清)、马血清、HAS(人血清白蛋白)、BSA(牛血清蛋白)。
优选地，所述谷氨酰胺可以更换为谷氨酰胺代替物。
优选地，所述谷氨酰胺代替物包括GlutaMAX TM Supplement。
更优选地，所述RDM4培养基是89％DMEM/F12，10％KSR，1％N2培养基，1％L-谷氨酰胺以及10mM Vitamin B3组成。
优选地，所述诱导视网膜色素上皮细胞成熟包括使用RMM培养基培养色素上皮前体细胞。
优选地，所述RMM培养基的组成包括DMEM/F12，B27培养基，谷氨酰胺。
优选地，所述RMM培养基由97％DMEM/F12，2％B27培养基，1％L-谷氨酰胺组成。
优选地，所述DMEM/F12或B27培养基还可以被William’s E细胞培养基、Neurobasal Medium细胞培养基、MEM细胞培养基、DMEM细胞培养基、1640RPMI细胞培养基，或F12细胞培养基等中的一种或多种的混合替代。
优选地，所述KSR还可以被其他血清类似物替代，所述其他血清类似物包括但不限于FBS(胎牛血清)、马血清、HAS(人血清白蛋白)、BSA(牛血清蛋白)。
优选地，所述谷氨酰胺可以更换为谷氨酰胺代替物。
优选地，所述谷氨酰胺代替物包括GlutaMAX TM Supplement。
优选地，所述视网膜色素上皮细胞传代包括使用REM培养基培养成熟的视网膜色素上皮细胞。
优选地，所述REM培养基的组成包括DMEM/F12，KSR，谷氨酰胺，β-巯基乙醇。
优选地，所述REM培养基由79％DMEM/F12，20％KSR，1％L-谷氨酰胺，50μMβ-巯基乙醇组成。
优选地，所述DMEM/F12还可以被William’s E细胞培养基、Neurobasal Medium细胞培养基、MEM细胞培养基、DMEM细胞培养基、1640RPMI细胞培养基，或F12细胞培养基等中的一种或多种的混合替代。
优选地，所述KSR还可以被其他血清类似物替代，所述其他血清类似物包括但不限于FBS(胎牛血清)、马血清、HAS(人血清白蛋白)、BSA(牛血清蛋白)。
优选地，所述谷氨酰胺可以更换为谷氨酰胺代替物。
优选地，所述谷氨酰胺代替物包括GlutaMAX TM Supplement。
优选地，所述β-巯基乙醇还可以被其他还原剂代替，所述还原剂包括但不限于β-巯基乙醇、二硫苏糖醇、二硫赤藓糖醇、还原型谷胱甘肽、半胱氨酸、硫代胺甲酸盐、二硫亚磺酸钠、抗坏血酸盐、二氯化锡或硼氢化钠。
优选地，本发明中所标注的浓度皆为终浓度，百分比皆为体积的百分比。
优选地，使用所述RDM1、RDM2、RDM3、RDM4、RMM或REM任一培养基时的换液频率根据细胞的生长状态进行调整；优选地，所述换液频率是每日换液。
优选地，使用所述RDM1培养基的总天数为3-9天。
优选地，使用所述RDM1培养基的总天数为6天。
优选地，使用所述RDM2培养基的总天数为2-8天。
优选地，使用所述RDM2培养基的总天数为5天。
优选地，使用所述RDM3培养基的总天数为1-7天。
优选地，使用所述RDM3培养基的总天数为4天。
优选地，使用所述RDM4培养基的总天数为3-9天。
优选地，使用所述RDM4培养基的总天数为6天。
优选地，使用所述RMM培养基的总天数为6-12天。
优选地，使用所述RMM培养基的总天数为9天。
优选地，所述方法中还包括细胞检测的步骤。
优选地，所述细胞检测可以是细胞活性检测、基于免疫的检测、流式细胞仪检测、比色检测、基于金纳米颗粒的检测、荧光检测、紫外检测、细胞标志物的检测中的一种或多种。
优选地，所述方法使用本领域技术人员通用的细胞培养方法处理细胞，所述细胞培养是利用培养基在体外进行的细胞制备、细胞分选、细胞克隆培养、细胞扩增培养、细胞富集、细胞纯化、细胞工程、细胞三维培养、细胞发酵、组织培养、器官培养的任一形式。
优选地，所述细胞培养可以在培养箱进行，也可以在其他适宜细胞生长的环境进行。
优选地，所述培养箱是CO 2培养箱。
优选地，所述培养箱是恒温培养箱；更优选的，恒温37℃。
第二方面，本发明提供了一种快速高效诱导RPE祖细胞的方法，所述方法包括使用含有小分子化合物的培养基进行培养干细胞。
所述小分子化合物包括BMP信号通路抑制剂，Wnt通路抑制剂，TGF-βI型受体ALK5、ALK4和ALK7的抑制剂，ROCK通路抑制剂，WNT信号通路激活剂，VEGFR激酶抑制剂中的任意一种或多种。
优选地，所述含有小分子化合物的培养基是前述RDM1培养基和/或RDM2培养基。
第三方面，本发明提供了一种诱导色素上皮前体细胞的方法，所述方法包括使用含有小分子化合物的培养基进行细胞培养。
所述小分子化合物包括GSK信号通路抑制剂，VEGFR激酶抑制剂，ROCK通路抑制剂，维生素或者维生素类似物中的任意一种或多种。
优选地，所述含有小分子化合物的培养基是权利要求3所述的RDM3培养基。
优选地，所述细胞培养是对RPE祖细胞所进行的培养。
优选地，所述RPE祖细胞是由前述快速高效诱导RPE祖细胞的方法制备得到的。
优选地，所述方法还包括使用前述RDM4培养基培养细胞。
第四方面，本发明提供了一种培养基，所述培养基选自以下任意一种：前述RDM1培养基、RDM2培养基、RDM3培养基、RDM4培养基、RMM培养基、REM培养基。
第五方面，本发明提供了一种培养基组合，所述培养基组合选自以下任意多种的组合：前述RDM1培养基、RDM2培养基、RDM3培养基、RDM4培养基、RMM培养基、REM培养基。
第六方面，本发明提供了诱导视网膜色素上皮细胞的试剂盒，所述试剂盒包括至少以下一种物质：BMP信号通路抑制剂，Wnt通路抑制剂，TGF-βI型受体ALK5、ALK4和ALK7的抑制剂，ROCK通路抑制剂，WNT信号通路激活剂，VEGFR激酶抑制剂，GSK信号通路抑制剂，VEGFR激酶抑制剂，维生素及其类似物。
或者，所述试剂盒包括配制前述RDM1培养基、RDM2培养基、RDM3培养基、RDM4培养基、RMM培养基、REM培养基中任意一种或多种的试剂。
优选地，本发明所述的RDM1培养基、RDM2培养基、RDM3培养基、RDM4培养基、RMM培养基、REM培养基可以是人为配置的培养基，也可以是商品化的培养基。
优选地，所述试剂盒中还包括培养细胞所需要的仪器。
优选地，所述仪器包括但不限于培养器皿(例如培养板、培养皿、培养瓶)、培养箱(包括CO 2培养箱)、生物安全柜、离心机、水浴仪器、冰箱、纯水设备、显微镜、干燥箱、细胞冷冻储存器、消毒器。
第七方面，本发明提供了前述RDM1培养基、RDM2培养基、RDM3培养基、RDM4培养基、RMM培养基、REM培养基、培养基组合、试剂盒、BMP信号通路抑制剂，Wnt通路抑制剂，TGF-βI型受体ALK5、ALK4和ALK7的抑制剂，ROCK通路抑制剂，WNT信号通路激活剂，VEGFR激酶抑制剂，GSK 信号通路抑制剂，维生素及其类似物中的任意一种在诱导视网膜色素上皮细胞中的应用。
第八方面，本发明提供了通过前述方法制备得到的视网膜色素上皮细胞及其在制备眼科疾病药物中的应用。
第九方面，本发明提供了一种治疗眼科疾病的方法，所述治疗眼科疾病的方法包括使用前述方法制备视网膜色素上皮细胞。
优选地，所述治疗眼科疾病的方法还包括进行细胞移植。
优选地，所述眼科疾病包括视网膜退行性疾病；所述视网膜退行性疾病主要病症包括视网膜色素上皮细胞的不可逆损失，并最终导致视觉功能丧失。
优选地，所述视网膜退行性疾病主要包括视网膜色素变性(retinitis pigmentosa，RP)、黄斑变性、Leber病(又名Leber先天性黑朦，Leber congenital amaurosis)、Usher综合征、视网膜萎缩(包括病变损害视网膜造成的或遗传因素引起的视网膜畏缩)。
优选地，所述黄斑变性包括少年黄斑变性(Stargarde，也称先天性黄斑变性)和年龄相关性黄斑变性(age related-macular degeneration，AMD)。
第十方面，本发明提供了以下应用：
1)BMP信号通路抑制剂，Wnt通路抑制剂，TGF-βI型受体ALK5、ALK4和ALK7的抑制剂，ROCK通路抑制剂，WNT信号通路激活剂，VEGFR激酶抑制剂，GSK信号通路抑制剂、维生素及其类似物中的任意一种在诱导视网膜色素上皮细胞中的应用；
2)BMP信号通路抑制剂，Wnt通路抑制剂，TGF-βI型受体ALK5、ALK4和ALK7的抑制剂，ROCK通路抑制剂，WNT信号通路激活剂，VEGFR激酶抑制剂，GSK信号通路抑制剂、维生素及其类似物中的任意一种在诱导视网膜色素上皮细胞中的应用；
3)RDM1培养基、RDM2培养基、BMP信号通路抑制剂，Wnt通路抑制剂，TGF-βI型受体ALK5、ALK4和ALK7的抑制剂，ROCK通路抑制剂，WNT信号通路激活剂，VEGFR激酶抑制剂，前述试剂盒在诱导RPE祖细胞中的应用；
4)RDM3培养基、RDM4培养基、GSK信号通路抑制剂，VEGFR激酶抑制剂，ROCK通路抑制剂，维生素或者维生素类似物、前述试剂盒在诱导色素上皮前体细胞中的应用。
第十一方面，本发明提供了以下细胞群：
1)由前述诱导RPE祖细胞的方法制备得到的细胞群，所述细胞群中表达PAX6和RPE65的细胞比例至少5％；优选地，至少10％；
2)由前述诱导色素上皮前体细胞的方法制备得到的细胞群，所述细胞群中表达PAX6和RPE65的细胞比例至少10％；优选地，至少20％；
3)由前述诱导视网膜色素上皮细胞的方法制备得到的细胞群，所述细胞群中表达ZO-1，RPE65，Pax6，CRALBP的阳性细胞比例至少80％；
4)由前述诱导视网膜色素上皮细胞的方法制备得到的细胞群，所述细胞群中TYRP2，PEDF，PMEL17，RPE65，CRALBP表达的水平相对于iPSC提高至少100倍。
附图说明
图1是第2天，第7天以及第12天在4倍光学显微镜下细胞形态变化；A：第2天，B：第7天，C：第14天。
图2是第14天，第18天以及第24天在4倍光学显微镜下细胞形态变化；A：第14天，B：第18天，C：第24天。
图3是第52天在4倍、10倍、20倍光学显微镜下细胞形态变化；A：4倍，B：10倍，C：20倍。
图4是第52天细胞肉眼下整体形态。
图5是第6天，第12天以及第24天PAX6和RPE65双阳性细胞检测；第一列是DAPI染色，第二列是PAX6染色，第三列是RPE65染色，第四列是PAX6和RPE65的融合。
图6是第12天RPE祖细胞相关基因的mRNA相对表达量统计结果图。
图7是第36天RPE细胞相关基因免疫荧光检测结果图；A是白光下的细胞形态，B是ZO1染色的结果图，C是PAX6和RPE65染色的结果图，D是ZO1和CRALBP染色的结果图。
图8是第36天RPE细胞相关基因的mRNA相对表达量统计结果图。
图9是使用不同的BMP抑制剂时，第6天细胞的PAX6的mRNA相对表达量统计结果图。
具体实施方式
下面结合实施例对本发明做进一步的说明，以下所述，仅是对本发明的较佳实施例而已，并非对本发明做其他形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容，依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化，均落在本发明的保护范围内。
通用方法1、免疫荧光检测步骤
1、在24孔培养板中将已爬好细胞的玻片用PBS浸洗3次，每次3min；
2、用4％的多聚甲醛室温固定爬片15min，PBS浸洗玻片3次，每次3min；
3、0.5％Triton X-100(5％BSA配制)室温通透20min(细胞膜上表达的抗原省略此步骤)；
4、使用5％的BSA室温封闭1小时；
5、去除封闭液，每孔添加400μl一抗，4度过夜孵育；
6、12小时后，加荧光二抗：PBST浸洗3次，每次5min，添加稀释好的荧光二抗，室温孵育1h，PBST浸洗3次，每次5min；
9、复染核：滴加DAPI避光孵育5min，对标本进行染核，PBST 5min×4次洗去 多余的DAPI；
10、添加500μl PBS，上机拍片。
通用方法2、qPCR检测基因表达水平
1、收集细胞200W细胞加1ml TRIZOL，提取RNA并测定RNA浓度，取1μg RNA反转为cDNA，按如下表1体系进行预混，
表1.PCR反应体系
Figure PCTCN2022107890-appb-000001
2、然后将上述体系放入Light cycler仪器中按照3步法进行反应，循环数为45，反应体系如下表2：
表2.PCR的反应程序
Figure PCTCN2022107890-appb-000002
实施例1、RPE分化流程及检测结果
本发明所使用的试剂如下表3所示：
表3.本发明所使用的试剂
名称 厂家 货号
DMEM/F-12 with HEPES GIBCO 11330032
matrigel Corning 354277
KnockOut Serum Replacement(KSR) GIBCO A3181502
L-谷氨酰胺(100X) GIBCO 35050061
LDN193189 sigma SML0559
LY2109761 abmole M2081
XAV-939 abmole M1796
Thiazovivin abmole M1856
6-bromoindirubin-3′-oxime(BIO) abmole M7627
SU 5402 biogems 2159233
Vitamin B3 sigma N3376-100G
N2 supplement GIBCO 17502001
B-27 TM Supplement,XenoFree,minus vitamin A Life technologies A3353501
0.25％Trypsin-EDTA(1X) Thermo 25200072
Chemically Defined Lipids GIBCO 0920239SA
Monothioglycerol Solution Sigma M6145
培养流程：
(1)诱导外胚层分化
1、第0天，将iPSC细胞以1.0×10 3-5.0×10 5/cm 2铺种，本案例以密度为5.0×10 3cells/cm 2铺种细胞，细胞培养在37℃/5％CO 2细胞培养箱中，使用培养基为RDM1。
RDM1的基础培养基由88％DMEM/F12，10％KSR，5mM Monothioglycerol Solution，1％Chemically Defined Lipid Concentrate，1％L-谷氨酰胺组成。
同时RDM1培养基中还含有：
1)BMP信号通路抑制剂：50ng/mL noggin
2)Wnt通路抑制剂：1μM的XAV-939；
3)TGF-βI型受体ALK5，ALK4和ALK7的抑制剂：5μM的LY2109761
4)ROCK通路抑制剂为10uM的Thiazovivin。
2、第1到第6天，每日更换RDM1培养基，到第6天，分化成功标志为至少5％的PAX6，RPE65双阳性细胞，检测方法见通用方法1，结果图如图5一行所示。
3、第7到第12天，每日更换RDM2培养基，RDM2的基础培养基由88％DMEM/F12，10％KSR，5mM Monothioglycerol Solution，1％Chemically Defined Lipid Concentrate，1％L-谷氨酰胺组成。
同时RDM2培养基中还含有：
1)WNT信号通路激活剂：10μM的6-bromoindirubin-3′-oxime(BIO)。
2)VEGFR激酶抑制剂：2μM的SU5402
3)ROCK通路抑制剂：10μM的Thiazovivin。
本实施例分化成功标志为产生至少10％PAX6和RPE65双阳性细胞，检测方法见通用方法1，检测结果如图5第二行所示；或者PAX6，RPE65，IHX2，pmel17的表达水平提高5倍，检测方法见通用方法2，结果如图6所示。
(2)诱导色素上皮前体细胞分化
4、第13到第17天，每日更换RDM3培养基，RDM3的基础培养基由88％DMEM/F12，10％KSR，5mM Monothioglycerol Solution，1％Chemically Defined Lipid Concentrate，1％L-谷氨酰胺组成。
同时RDM3培养基中还含有：
1)GSK信号通路抑制剂：10μM的6-bromoindirubin-3′-oxime(BIO)；
2)VEGFR激酶抑制剂：2μM的SU5402；
3)ROCK通路抑制剂：10μM的Thiazovivin；
4)维生素及其类似物；10mM的Vitamin B3。
5、第18到第24天，每日更换RDM4培养基，RDM4的基础培养基由89％DMEM/F12，10％KSR，1％N2培养基，1％L-谷氨酰胺以及10mM Vitamin B3组成。
本实施例分化成功标志为产生至少20％PAX6和RPE65阳性细胞，检测方法见通用方法1，检测结果如图5第三行所示。
(3)诱导视网膜色素上皮细胞成熟
6、第25到第36天，每日更换RMM培养基，RMM的基础培养基由97％DMEM/F12，2％B27培养基，1％L-谷氨酰胺组成。
本实施例分化成功标志为产生至少80％ZO-1，RPE65，Pax6，CRALBP的阳性细胞，不少于1％的细胞产生黑色素沉着。检测方法见通用方法1，检测结果如图7所示；或者qPCR检测TYRP2，PEDF，PMEL17，RPE65，CRALBP表达水平提高至少100倍，检测方法见通用方法2，检测结果如图8所示。
(4)视网膜色素上皮细胞传代
7、第37天以后，吸去旧的培养基，用室温DPBS洗两遍，随后加入1mL 37℃预热过的0.25％Trypsin–EDTA，置于37℃/5％CO 2细胞培养箱中10min，显微镜下观察单个细胞间出现空隙。
8、弃去Trypsin–EDTA，加入3ml的REM培养基终止消化；
9、使用35μM过滤器过滤后，转移至15ml离心管，室温1000rpm离心5min。
10、弃去上清，用REM培养基轻轻吹打细胞然后重悬。计数后铺板至matrigel包被的六孔板中。
11、第38-51天隔天更换一次REM，直至收集细胞冻存。
本实施例中REM培养基由79％DMEM/F12，20％KSR，1％L-谷氨酰胺，50μMβ巯基乙醇组成。
检测结果：
在4倍光学显微镜下观察细胞形态变化，第2天，第7天以及第12天的细胞形态如图1。第2天细胞呈现集落生长，第7天细胞扩增，呈现明显上皮样形态，第12天部分上皮样细胞开始堆积生长。第14天，第18天以及第24天的细胞形态如图2。第14天上皮样细胞呈现集落堆积生长，第24天上皮样细胞呈现集落堆积生长，细胞形态改变，细胞边界更加分明，第24天上皮样细胞呈现集落堆积生长，细胞形态呈现不规则多边形。
第52天在4倍、10倍、20倍光学显微镜下细胞形态变化，结果如图3所示。4倍、10倍镜下，上皮样细胞呈现集落堆积生长，细胞形态改变，细胞边界更加分明，20倍光学显微镜下，细胞呈现典型的正六边形形态。第52天细胞肉眼下整体形态如图4。80％左右细胞在肉眼下呈现黑色色素沉积。
图5是第6天，第12天以及第24天PAX6和RPE65双阳性细胞检测。第6天，成功分化产生至少5％的PAX6和RPE65双阳性细胞；第12天，成功分化产生至少10％PAX6和RPE65双阳性细胞；第24天，成功分化产生至少20％PAX6和RPE65双阳性细胞。
图6是第12天RPE祖细胞相关基因检测。PAX6，RPE65，IHX2，pmel17的表达水平相比iPSC提高至少5倍。OCT4和NANOG均为iPSC标记基因，RPE祖细胞表达较低。
图7是第36天RPE细胞相关基因检测。分化产生至少80％的ZO-1，RPE65，Pax6，CRALBP的阳性细胞
图8是第36天RPE细胞相关基因检测。TYRP2，PEDF，PMEL17，RPE65，CRALBP表达水平相对于iPSC提高至少100倍。
实施例2、RDM1培养基中使用不同的BMP信号通路抑制剂进行细胞诱导
按照实施例1的方法对iPSC细胞进行培养，仅在RDM1培养基中加入50ng/mL noggin或者2μM的Dorsomorphin或者3μM的LDN-193189作为3组平行对照，对第6天细胞的PAX6表达水平进行检测。
结果如图9所示：Day0天为对照，细胞分化6天后检测PAX6的表达水平，其中使用noggin效果最好，PAX6表达水平最高。

Claims (12)

  1. 一种RDM1培养基，其特征在于，所述RDM1培养基包括BMP信号通路抑制剂，Wnt通路抑制剂，TGF-βI型受体ALK5、ALK4和ALK7的抑制剂，ROCK通路抑制剂中的至少一种；
    优选地，所述BMP信号通路抑制剂包括noggin、Dorsomorphin、DMH1、LDN-193189；
    优选地，所述BMP信号通路抑制剂包括50-200ng/mL的noggin、2-8μM的Dorsomorphin、10-100μM的DMH1、5nM-5μM的LDN-193189；
    优选地，所述BMP信号通路抑制剂包括50ng/mL的noggin、2μM的Dorsomorphin、3μM的LDN-193189；
    优选地，所述BMP信号通路抑制剂选用noggin；
    优选地，所述noggin的工作浓度为50ng/mL；
    优选地，所述Wnt通路抑制剂包括XAV-939、iCRT-3、iCRT-5、iCRT-14、IWP-4、IWR-1、wnt-C59；
    优选地，所述Wnt通路抑制剂为2-20μM的XAV-939；
    优选地，所述TGF-βI型受体ALK5、ALK4和ALK7的抑制剂包括LY2109761、A83-01、SB-525334、SD-208、EW-7197、Disitertide、LY3200882、SM16、SB431542；
    优选地，所述TGF-βI型受体ALK5、ALK4和ALK7的抑制剂是2-20μM的LY2109761；
    优选地，所述TGF-βI型受体ALK5、ALK4和ALK7的抑制剂是5μM的LY2109761；
    优选地，所述ROCK通路抑制剂包括Thiazovivin、Y-27632；
    优选地，所述ROCK通路抑制剂是0.5-20μM的Thiazovivin；
    优选地，所述ROCK通路抑制剂是10μM的Thiazovivin；
    优选地，所述RDM1培养基的基础培养基是RDM基础培养基，所述RDM基础培养基是由DMEM/F12，KSR，Monothioglycerol Solution，Chemically Defined Lipid Concentrate，谷氨酰胺组成；
    优选地，所述DMEM/F12替代为William’s E细胞培养基、Neurobasal Medium细胞培养基、MEM细胞培养基、DMEM细胞培养基、1640RPMI细胞培养基，或F12细胞培养基等中的一种或多种；
    优选地，所述KSR替代为其他血清类似物替代，所述其他血清类似物包括FBS、马血清、HAS、BSA；
    优选地，所述谷氨酰胺替代为GlutaMAX TMSupplement或L-谷氨酰胺；
    优选地，所述RDM基础培养基是由88％DMEM/F12，10％KSR，5mM Monothioglycerol Solution，1％Chemically Defined Lipid Concentrate，1％L-谷氨酰胺组成。
  2. 一种RDM2培养基，其特征在于，所述培养基包括WNT信号通路激活剂，VEGFR激酶抑制剂、ROCK通路抑制剂中的至少一种；
    优选地，所述WNT信号通路激活剂包括6-bromoindirubin-3′-oxime；
    优选地，所述WNT信号通路激活剂为1-20μM的6-bromoindirubin-3′-oxime；
    优选地，所述WNT信号通路激活剂为10μM的6-bromoindirubin-3′-oxime；
    优选地，所述VEGFR激酶抑制剂包括SU5402、AV-951、SU5205、SU5408；
    优选地，所述VEGFR激酶抑制剂为1-20μM的SU5402；
    优选地，所述VEGFR激酶抑制剂为2μM的SU5402；
    优选地，所述ROCK通路抑制剂包括Thiazovivin、Y-27632；
    优选地，所述ROCK通路抑制剂为0.5-20μM的Thiazovivin；
    优选地，所述ROCK通路抑制剂为10μM的Thiazovivin；
    优选地，所述RDM1培养基的基础培养基是RDM基础培养基，所述RDM基础培养基是由DMEM/F12，KSR，Monothioglycerol Solution，Chemically Defined Lipid Concentrate，谷氨酰胺组成；
    优选地，所述DMEM/F12替代为William’s E细胞培养基、Neurobasal Medium细胞培养基、MEM细胞培养基、DMEM细胞培养基、1640RPMI细胞培养基，或F12细胞培养基等中的一种或多种；
    优选地，所述KSR替代为其他血清类似物替代，所述其他血清类似物包括FBS、马血清、HAS、BSA；
    优选地，所述谷氨酰胺替代为GlutaMAX TMSupplement或L-谷氨酰胺；
    优选地，所述RDM基础培养基是由88％DMEM/F12，10％KSR，5mM Monothioglycerol Solution，1％Chemically Defined Lipid Concentrate，1％L-谷氨酰胺组成。
  3. 一种RDM3培养基，其特征在于，所述培养基包括GSK信号通路抑制剂，VEGFR激酶抑制剂，ROCK通路抑制剂，维生素或者维生素类似物中的至少一种；
    优选地，所述GSK信号通路抑制剂包括6-bromoindirubin-3′-oxime；
    优选地，所述GSK信号通路抑制剂为1-20μM的6-bromoindirubin-3′-oxime；
    优选地，所述GSK信号通路抑制剂为10μM的6-bromoindirubin-3′-oxime；
    优选地，所述VEGFR激酶抑制剂包括SU5402、AV-951、SU5205、SU5408；
    优选地，所述VEGFR激酶抑制剂为1-20μM的SU5402；
    优选地，所述VEGFR激酶抑制剂为2μM的SU5402；
    优选地，所述ROCK通路抑制剂包括Thiazovivin、Y-27632；
    优选地，所述ROCK通路抑制剂为0.5-20μM的Thiazovivin；
    优选地，所述ROCK通路抑制剂为10μM的Thiazovivin；
    优选地，所述维生素或者维生素类似物包括生物素、氯化胆碱、D-泛酸钙、叶酸、肌醇、烟酰胺、吡哆醇盐酸盐、核黄素、盐酸石克氨、辅酶Q10、腐胺二盐酸盐、维生素A、Vitamin B、维生素C、维生素D、维生素E、维生素K、维生素H、维生素P、维生素M、维生素T、维生素U、水溶性维生素；
    优选地，所述维生素或者维生素类似物包括Vitamin B；
    优选地，所述维生素或者维生素类似物是Vitamin B3；
    优选地，所述维生素或者维生素类似物是1-20mM的Vitamin B3；
    优选地，所述维生素或者维生素类似物是10mM的Vitamin B3；
    优选地，所述RDM3培养基的基础培养基是RDM基础培养基，所述RDM基础培养基是由DMEM/F12，KSR，Monothioglycerol Solution，Chemically Defined Lipid Concentrate，谷氨酰胺组成；
    优选地，所述DMEM/F12替代为William’s E细胞培养基、Neurobasal Medium细胞培养基、MEM细胞培养基、DMEM细胞培养基、1640RPMI细胞培养基，或F12细胞培养基等中的一种或多种；
    优选地，所述KSR替代为其他血清类似物替代，所述其他血清类似物包括FBS、马血清、HAS、BSA；
    优选地，所述谷氨酰胺替代为GlutaMAX TMSupplement或L-谷氨酰胺；
    优选地，所述RDM基础培养基是由88％DMEM/F12，10％KSR，5mM Monothioglycerol Solution，1％Chemically Defined Lipid Concentrate，1％L-谷氨酰胺组成。
  4. 一种快速高效诱导RPE祖细胞的方法，所述方法包括使用含有小分子化合物的培养基进行培养干细胞；
    所述小分子化合物包括BMP信号通路抑制剂，Wnt通路抑制剂，TGF-βI型受体ALK5、ALK4和ALK7的抑制剂，ROCK通路抑制剂，WNT信号通路激活剂，VEGFR激酶抑制剂中的任意一种或多种；
    优选地，所述含有小分子化合物的培养基是权利要求1所述的RDM1培养基和/或权利要求2所述的RDM2培养基；
    优选地，所述方法包括使用RDM1培养基培养细胞；
    优选地，经过所述RDM1培养基的培养所述细胞中表达PAX6和RPE65的细胞比例增加；
    优选地，经过所述RDM1培养基的培养所述细胞中表达PAX6和RPE65的细胞比例增加5％；
    优选地，经过所述RDM1培养基的培养所述细胞中表达PAX6和RPE65的细胞比例增加到至少5％；
    优选地，所述方法包括使用RDM2培养基培养细胞；
    优选地，经过所述RDM2培养基的培养所述细胞中表达PAX6和RPE65的细胞比例增加；
    优选地，经过所述RDM2培养基的培养所述细胞中表达PAX6和RPE65的细胞比例增加5％；
    优选地，经过所述RDM2培养基的培养所述细胞中表达PAX6和RPE65的细胞比例增加从5％到至少10％；
    优选地，经过所述RDM1培养基和/或RDM2培养基的培养的细胞中PAX6、RPE65、IHX2、pmel17的表达量提高。
  5. 如权利要求4所述的方法，其特征在于，所述干细胞包括全能干细胞、多能干细胞、单能干细胞；
    优选地，所述干细胞是多能干细胞；
    优选地，所述干细胞是诱导性多能干细胞；
    优选地，所述干细胞是人源的iPSC细胞。
  6. 一种诱导色素上皮前体细胞的方法，所述方法包括使用含有小分子化合物的培养基进行细胞培养；
    所述小分子化合物包括GSK信号通路抑制剂，VEGFR激酶抑制剂，ROCK通路抑制剂，维生素或者维生素类似物中的任意一种或多种；
    优选地，所述含有小分子化合物的培养基是权利要求3所述的RDM3培养基；
    优选地，经过所述RDM3培养基的培养所述细胞中表达PAX6和RPE65的细胞比例增加；
    优选地，经过所述RDM3培养基的培养所述细胞中表达PAX6和RPE65的细胞比例增加10％；
    优选地，经过所述RDM3培养基的培养所述细胞中表达PAX6和RPE65的细胞比例增加到至少20％。
  7. 如权利要求6所述的方法，其特征在于，所述细胞培养是对RPE祖细胞所进行的培养；
    优选地，所述RPE祖细胞是由权利要求4所述的方法制备得到的；
    优选地，所述方法还包括使用RDM4培养基培养细胞；所述RDM4培养基是DMEM/F12，KSR，N2培养基，谷氨酰胺以及维生素组成；
    优选地，所述RDM4培养基培养的是经过了RDM3培养基培养的细胞；
    优选地，所述DMEM/F12替代为William’s E细胞培养基、Neurobasal Medium细胞培养基、MEM细胞培养基、DMEM细胞培养基、1640RPMI细胞培养基，或F12细胞培养基等中的一种或多种；
    优选地，所述KSR替代为其他血清类似物替代，所述其他血清类似物包括FBS、马血清、HAS、BSA；
    优选地，所述谷氨酰胺替代为GlutaMAX TMSupplement或L-谷氨酰胺；
    优选地，所述RDM4培养基是89％DMEM/F12，10％KSR，1％N2培养基，1％L-谷氨酰胺以及10mM Vitamin B3组成。
  8. 一种诱导视网膜色素上皮细胞的方法，其特征在于，所述方法包括以下步骤中的至少一种：
    1)诱导RPE祖细胞的步骤；
    2)诱导色素上皮前体细胞的步骤；
    3)诱导色素上皮细胞成熟的步骤；
    4)色素上皮细胞传代培养的步骤；
    优选地，所述诱导RPE祖细胞的步骤是使用权利要求4所述的方法；
    优选地，所述诱导色素上皮前体细胞的步骤是使用权利要求6所述的方法；
    优选地，所述诱导视网膜色素上皮细胞成熟包括使用RMM培养基培养色素上皮前体细胞；
    优选地，所述视网膜色素上皮细胞传代包括使用REM培养基培养成熟的视网膜色素上皮细胞。
  9. 如权利要求8所述的方法，其特征在于，所述RMM培养基的组成包括DMEM/F12，B27培养基，谷氨酰胺；所述REM培养基的组成包括DMEM/F12，KSR，谷氨酰胺，β-巯基乙醇；
    优选地，所述DMEM/F12替代为William’s E细胞培养基、Neurobasal Medium细胞培养基、MEM细胞培养基、DMEM细胞培养基、1640RPMI细胞培养基，或F12细胞培养基等中的一种或多种；
    优选地，所述KSR替代为其他血清类似物替代，所述其他血清类似物包括FBS、马血清、HAS、BSA；
    优选地，所述谷氨酰胺替代为GlutaMAX TMSupplement或L-谷氨酰胺；
    优选地，所述β-巯基乙醇替代为其他还原剂代替，所述还原剂包括但不限于β-巯基乙醇、二硫苏糖醇、二硫赤藓糖醇、还原型谷胱甘肽、半胱氨酸、硫代胺甲酸盐、二硫亚磺酸钠、抗坏血酸盐、二氯化锡或硼氢化钠；
    优选地，所述RMM培养基由97％DMEM/F12，2％B27培养基，1％L-谷氨酰胺组成；
    优选地，所述REM培养基由79％DMEM/F12，20％KSR，1％L-谷氨酰胺，50μMβ-巯基乙醇组成。
  10. 一种试剂盒，所述试剂盒中包括至少以下一种物质：
    1)权利要求1所述的BMP信号通路抑制剂，
    2)权利要求1所述的Wnt通路抑制剂，
    3)权利要求1所述的TGF-βI型受体ALK5、ALK4和ALK7的抑制剂，
    4)权利要求1或2或3所述的ROCK通路抑制剂，
    5)权利要求2所述的WNT信号通路激活剂，
    6)权利要求2或3所述的VEGFR激酶抑制剂，
    7)权利要求3所述的GSK信号通路抑制剂，
    8)权利要求3所述的维生素及其类似物。
  11. 一种应用，其特征在于，所述应用选自以下任意一种：
    1)权利要求1所述的RDM1培养基、权利要求2所述的RDM2培养基、权利要求3所述的RDM3培养基、权利要求7所述的RDM4培养基、权利要求8所述的RMM培养基、权利要求8所述的REM培养基、权利要求10所述的试剂盒中的任意一种在诱导视网膜色素上皮细胞中的应用；
    2)BMP信号通路抑制剂，Wnt通路抑制剂，TGF-βI型受体ALK5、ALK4和ALK7的抑制剂，ROCK通路抑制剂，WNT信号通路激活剂，VEGFR激酶抑制剂，GSK信号通路抑制剂、维生素及其类似物中的任意一种在诱导视网膜色素上皮细胞中的应用；
    权利要求1所述的BMP信号通路抑制剂，权利要求1所述的Wnt通路抑制剂，权利要求1所述的TGF-βI型受体ALK5、ALK4和ALK7的抑制剂，权利要求1或2或3所述的ROCK通路抑制剂，权利要求2所述的WNT信号通路激活剂，权利要求2或3所述的VEGFR激酶抑制剂，权利要求3所述的GSK信号通路抑制剂、权利要求3所述的维生素及其类似物中的任意一种在诱导视网膜色素上皮细胞中的应用；
    3)权利要求1所述的RDM1培养基、权利要求2所述的RDM2培养基、BMP信号通路抑制剂，Wnt通路抑制剂，TGF-βI型受体ALK5、ALK4和ALK7的抑制剂，ROCK通路抑制剂，WNT信号通路激活剂，VEGFR激酶抑制剂，权利要求10所述的试剂盒在诱导RPE祖细胞中的应用；
    优选地，权利要求1所述的RDM1培养基、权利要求2所述的RDM2培养基、权利要求1所述的BMP信号通路抑制剂，权利要求1所述的Wnt通路抑制剂，权利要求1所述的TGF-βI型受体ALK5、ALK4和ALK7的抑制剂，权利要求1或2所述的ROCK通路抑制剂，权利要求2所述的WNT信号通路激活剂，权利要求2所述的VEGFR激酶抑制剂，权利要求10所述的试剂盒在诱导RPE祖细胞中的应用；
    4)权利要求3所述的RDM3培养基、权利要求7所述的RDM4培养基、GSK信号通路抑制剂，VEGFR激酶抑制剂，ROCK通路抑制剂，维生素或者维生素类似物、权利要求10所述的试剂盒在诱导色素上皮前体细胞中的应用；
    优选地，权利要求3所述的RDM3培养基、权利要求7所述的RDM4培养基、权利要求3所述的GSK信号通路抑制剂，权利要求3所述的ROCK通路抑制剂，权利要求3所述的VEGFR激酶抑制剂，权利要求3所述的维生素及其类似物、权利要求10所述的试剂盒在诱导色素上皮前体细胞中的应用；
    5)权利要求4-9任一所述的方法制备得到的细胞在制备眼科疾病药物中的应用；
    优选地，所述眼科疾病包括视网膜退行性疾病；
    优选地，所述视网膜退行性疾病主要包括视网膜色素变性、黄斑变性、Leber病、Usher综合征、视网膜萎缩；
    优选地，所述黄斑变性包括少年黄斑变性和年龄相关性黄斑变性。
  12. 一种细胞群，其特征在于，所述细胞群选自以下任意一种：
    1)由权利要求4所述方法制备得到的细胞群，所述细胞群中表达PAX6和RPE65的细胞比例至少5％；优选地，至少10％；
    2)由权利要求6所述方法制备得到的细胞群，所述细胞群中表达PAX6和RPE65的细胞比例至少10％；优选地，至少20％；
    3)由权利要求8所述方法制备得到的细胞群，所述细胞群中表达ZO-1，RPE65，Pax6，CRALBP的阳性细胞比例至少80％；
    4)由权利要求8所述方法制备得到的细胞群，所述细胞群中TYRP2，PEDF，PMEL17，RPE65，CRALBP表达的水平相对于iPSC提高至少100倍。
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