WO2022134031A1 - 一种光感受器神经元细胞的化学诱导方法 - Google Patents

一种光感受器神经元细胞的化学诱导方法 Download PDF

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WO2022134031A1
WO2022134031A1 PCT/CN2020/139521 CN2020139521W WO2022134031A1 WO 2022134031 A1 WO2022134031 A1 WO 2022134031A1 CN 2020139521 W CN2020139521 W CN 2020139521W WO 2022134031 A1 WO2022134031 A1 WO 2022134031A1
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cells
medium
concentration
stem cells
pluripotent stem
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French (fr)
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魏君
蔡萌
牛璐萌
周佳
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武汉睿健医药科技有限公司
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Priority to PCT/CN2020/139521 priority Critical patent/WO2022134031A1/zh
Priority to EP20966596.7A priority patent/EP4269564A1/en
Priority to US18/259,238 priority patent/US20240052305A1/en
Publication of WO2022134031A1 publication Critical patent/WO2022134031A1/zh

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Definitions

  • the present invention belongs to the biological field. It specifically relates to the chemical induction and culture method of photoreceptor neuron cells, the culture medium and its application in various diseases.
  • the ectoderm is the outermost layer formed during embryonic development. With the onset of organogenesis, ectoderm cells gradually differentiate into important systems such as the brain, spinal cord, and sensory organs.
  • the nervous system is an important system responsible for functions such as thinking, emotion, perception, and movement. Compared with diseases such as tumors, there are currently fewer drugs for neurological diseases and the development cycle is long. One of the most important reasons is the particularity of various primary cells in the ectodermal lineage, such as the non-regeneration of primary neurons. Sexuality has led to the scarcity of in vitro drug screening platforms for nervous system drugs.
  • the in vitro regeneration of ectodermal cells can solve a variety of degenerative diseases. For example, neurodegenerative diseases are common aging diseases.
  • Neurodegenerative diseases include amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD) and other diseases.
  • ALS amyotrophic lateral sclerosis
  • PD Parkinson's disease
  • AD Alzheimer's disease
  • ALS amyotrophic lateral sclerosis
  • PD Parkinson's disease
  • AD Alzheimer's disease
  • the difficulty in the treatment of such diseases lies in the non-regeneration of nerve cells in the central nervous system, and such diseases are caused by irreversible damage to the central nervous system.
  • nerve cell transplantation is the most effective treatment.
  • Degenerative diseases of the optic nerve are also a large category of diseases. Take age-related macular degeneration (AMD), the leading cause of vision impairment in people over the age of 50, characterized by retinal degeneration that can lead to loss of central vision.
  • AMD age-related macular degeneration
  • pigmentary degeneration a rare genetic disorder characterized by the progressive deterioration of photoreceptors in the retina, is unable to convert light signals into electrical signals to the brain.
  • the retina is an important tissue that can sense "images" in higher animals, and is composed of light-sensitive cells and photoreceptors. Photoreceptors can convert light signals from images into neural signals, and are important cells that allow the brain to "see” images.
  • cone receptor cells are responsible for our daytime vision and color perception, while rods are more sensitive and can perceive dim light even in low-light conditions.
  • the damage of photoreceptor cells is an important cause of retinal degenerative diseases, resulting in the failure of the eyeball to transmit the nerve signals converted from "images" to the brain, which seriously affects people's vision and even leads to blindness.
  • damage to photoreceptor nerve cells is an irreversible process that cannot repair itself, so there is no good treatment for such diseases.
  • the device consists of implants fixed under the retina and electrode compounds that stimulate retinal neurons to transmit information to the brain, but current artificial retinas are expensive and cannot restore visual function finely;
  • gene therapy based on viral vectors, which restores the function of inherited photoreceptors by correcting genetic mutations.
  • gene therapy in addition to being expensive, cannot solve the degenerative diseases of the optic nerve caused by age.
  • Yamanaka Shinya's team invented a "cocktail" method composed of four transcription factors OCT4, SOX2, KLF4 and c-Myc, which can successfully reprogram terminally differentiated skin fibroblasts into differentiated pluripotent cells.
  • Such stem cells are called induced pluripotent stem cells (Induced Pluripotent Cells) (Cell, 2006, 124(4) pp.663-676; Cell, 2007, 131(5) pp.861-872).
  • These stem cells have a similar differentiation potential to embryonic stem cells (Embryonic Stem Cells), and can form the three most basic germ layers in human development: ectoderm, mesoderm and endoderm, and eventually form a variety of adult cells.
  • This invention breaks through the ethical limitations of using human embryonic stem cells in medicine, can solve the problem of immune rejection in cell transplantation therapy, and greatly expand the application potential of stem cell technology in clinical medicine.
  • the use of totipotent stem cells or pluripotent stem cells including embryonic stem cells and iPSCs as raw materials to induce differentiation of ectodermal cells can be a new idea for clinical treatment, which greatly expands the application potential of ectodermal cells in clinical medicine.
  • the induction of neural stem cells and neurons mostly adopts the dual SMAD inhibition method (Nat Biotechnol, 2009, 05; 27(5): 485), which obtains
  • the neural stem cells can differentiate into other types of neuron cells, and the principle is to induce the generation of neural stem cells by inhibiting the BMP and TGFBeta pathways to mimic the signaling pathways in early embryonic development.
  • LDN-193189 and SB431542 are two widely used chemical small-molecule inhibitors.
  • ALK2 and ALK3 in the BMP4 pathway act on ALK2 and ALK3 in the BMP4 pathway, and ALK5 in the TGFBeta pathway, respectively, to achieve the formation of transplanted endoderm and mesoderm, thereby playing a role in the formation of transplanted endoderm and mesoderm.
  • the role of neurogenesis By this method, photoreceptor neurons can be obtained in the presence of serum-based components (Knockout Serum Replacement), fetal bovine serum (FBS) and retinoic acid, and the production cycle takes at least 70 days (STEM CELLS TRANSLATIONAL MEDICINE, 2018; 7:210-219; Sci Transl Med, 2019.1 16;11(475)).
  • retinoic acid is an important participating component of the visual cycle, involved in the conversion of vitamin A (known as all-trans retinol or all-trans ROL) to 11-cis-retinal ( 11-cis-RAL), transports 11-cis-RAL into photoreceptors, produces photopigment, isomerizes and reduces 11-cis-RAL to all-trans ROL, and transports all-trans ROL back to RPE Involved in another cycle (Nature. 1960; 168: 114-118; Nat. Genet. 1997; 15: 236-246; J. Biol. Chem. 1997; 272: 10303-10310).
  • Retinoids in cell culture systems such as retinoic acid (RA) are very susceptible to oxidation and become inactive. Therefore, in order to stabilize retinoic acid from rapid oxidative damage, serum is used in large quantities to stabilize retinoic acid in cell culture systems (Int J Dev Biol. 2012;56(4):273-8). Therefore, a retinoid chemical molecule that is neither serum-independent and stable under cell culture conditions is extremely important for the stable and safe induction of photoreceptor neurons in vitro.
  • RA retinoic acid
  • Galunisertib (LY2157299) used in the present invention, standard chemical name: 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl) quinoline-6-carboxamide or 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline -6-Carboxamide; is a small molecule inhibitor of TGF- ⁇ receptor I (TGF- ⁇ RI, ALK5).
  • LY2157299 is currently in Phase II clinical evaluation, with anticancer activity against liver cancer and glioblastoma (Giannelli G1, Villa E, Lahn M. Transforming Growth Factor- ⁇ as a Therapeutic Target in Hepatocellular Carcinoma. Cancer Res. 2014.4.1; 74(7):1890-4), inhibits tumor growth, invasion and metastasis by blocking the TGF- ⁇ signaling pathway; in addition, multiple studies have shown that LY2157299 blocks the production of CTGF and inhibits new angiogenesis, thereby inhibiting growth of cancer cells.
  • the development direction of this molecule is mainly based on the drug development and treatment of lung cancer, liver cancer and glioblastoma (Pharmaceutics. 2020.5.18;12(5):459), and has not disclosed any application in the field of nerve regeneration.
  • the present invention uses a serum-free small molecule chemical induction system to rapidly transform pluripotent stem cells into photoreceptor nerve cells.
  • the replacement of retinol by small molecules ensures the operation of the visual cycle required for the development of photoreceptor neurons and eliminates the adverse factors of serum. Therefore, the present invention not only completely eliminates the potential dangers posed by animal-derived components in the cell culture process, but also the combination of novel chemical small molecules exhibits great purity and yield advantages. Therefore, the present invention greatly expands the clinical prospect of photoreceptor nerve cell transplantation. At the same time, it solves the problems of low purity and long cycle in the production process of similar cell products, so it has huge economic and social effects.
  • the invention relates to a chemical induction and culture method of photoreceptor neuron cells, a culture medium and its application in various diseases.
  • the present invention relates to the following aspects:
  • the invention provides the use of LY2157299 in the differentiation of (eg, human-derived) pluripotent stem cells into ectoderm.
  • the present invention provides a medium for differentiation from (eg, human-derived) pluripotent stem cells into (eg, human-derived) ectoderm.
  • the medium for differentiating (eg, human-derived) pluripotent stem cells into (eg, human-derived) ectoderm is characterized in that LY2157299 is added to the NouvNeu-basal basal medium, the NouvNeu-basal basal medium
  • the formula is Duchenne's modified Eagle/F12 medium (DMEM/F12 medium), 0.1%-2% minimum essential medium non-essential amino acids (Minimum Essential Medium Non-Essential Amino Acids, the optimal concentration is 1%), 0.1-0.8g/L sodium chloride (Sodium Chloride, the optimum concentration is 0.5g/L), 0.1-80mg/L vitamin C (L-ascorbic acid, the optimum concentration is 64mg/L), 0.1-1.8uM vitamin B12 (optimal concentration is 1.2uM), 0.1-9.8ng/ml progesterone (Progesterone, optimum concentration is 6.3ng/ml), 10-35ug/ml putrescine (Putres
  • the dosage of LY2157299 is 55nM-25uM, preferably 100nM, 300nM, 500nM, 700nM, 900nM, 1uM, 2.5uM, 5uM, 7.5uM, 10uM, 12.5uM, 15uM, 17.5uM, 20uM or 22uM.
  • the dosage of the LY2157299 is 7.5uM.
  • the present invention provides a method for differentiating (eg, human-derived) pluripotent stem cells into (eg, human-derived) ectoderm, characterized in that the pluripotent stem cells are added with 55nM-25uM LY2157299 (preferably 100nM, 300nM, 500nM, 700nM, 900nM, 1uM, 2.5uM, 5uM, 7.5uM, 10uM, 12.5uM, 15uM, 17.5uM, 20uM or 22uM) in NouvNeu-basal basal medium to obtain ectodermal cells, the NouvNeu-basal
  • the formula of the basal medium is Duchenne's modified Eagle/F12 medium (DMEM/F12 medium), 0.1%-2% minimum essential medium non-essential amino acids (Minimum Essential Medium Non-Essential Amino Acids, the optimal concentration is 1 %), 0.1-0.8g/L sodium chloride (Sodium Chloride, the optimum concentration is 0.5g/
  • the present invention provides the use of LY2157299 and JW55 in the differentiation of (eg, human-derived) pluripotent stem cells into neural stem cells.
  • the present invention provides a medium for differentiating (eg, human-derived) pluripotent stem cells into neural stem cells, characterized in that LY2157299 and JW55 are added to a NouvNeu-basal basal medium, the NouvNeu-basal basal medium having The formula is Duchenne's modified Eagle/F12 medium (DMEM/F12 medium), 0.1%-2% minimum essential medium non-essential amino acids (Minimum Essential Medium Non-Essential Amino Acids, the optimal concentration is 1%), 0.1% -0.8g/L sodium chloride (Sodium Chloride, optimal concentration is 0.5g/L), 0.1-80mg/L vitamin C (L-ascorbic acid, optimal concentration is 64mg/L), 0.1-1.8uM vitamin B12 (the optimal concentration is 1.2uM), 0.1-9.8ng/ml progesterone (Progesterone, the optimal concentration is 6.3ng/ml), 10-35ug/ml putrescine (Putrescine,
  • the present invention provides a medium for differentiating (eg, human-derived) ectoderm into neural stem cells, characterized in that LY2157299 and JW55 are added to NouvNeu-basal basal medium, the formulation of which is NouvNeu-basal basal medium It is Duchenne's modified Eagle/F12 medium (DMEM/F12 medium), 0.1%-2% minimum essential medium non-essential amino acids (Minimum Essential Medium Non-Essential Amino Acids, the optimal concentration is 1%), 0.1- 0.8g/L sodium chloride (Sodium Chloride, the optimum concentration is 0.5g/L), 0.1-80mg/L vitamin C (L-ascorbic acid, the optimum concentration is 64mg/L), 0.1-1.8uM vitamin B12 ( The optimum concentration is 1.2uM), 0.1-9.8ng/ml progesterone (Progesterone, the optimum concentration is 6.3ng/ml), 10-35ug/ml putrescine (
  • the present invention provides a method for differentiating (eg, human-derived) pluripotent stem cells into neural stem cells, characterized by culturing with the aforementioned medium for differentiating (eg, human-derived) pluripotent stem cells into neural stem cells.
  • the present invention provides a method for differentiating (eg, human-derived) ectodermal cells into neural stem cells, characterized by culturing with the aforementioned medium for differentiating (eg, human-derived) ectodermal cells into neural stem cells.
  • the present invention provides the use of LY2157299 and AM580 in the differentiation of (eg, human-derived) pluripotent stem cells into photoreceptor neuron cells.
  • the dosage of LY2157299 is 55nM-25uM, preferably 100nM, 300nM, 500nM, 700nM, 900nM, 1uM, 2.5uM, 5uM, 7.5uM, 10uM, 12.5uM, 15uM, 17.5uM, 20uM or 22uM.
  • the dosage of AM580 is 0.05-0.95uM, preferably 0.08uM, 0.09uM, 0.1uM, 0.2uM, 0.3uM, 0.5uM, 0.6uM, 0.7uM, 0.8uM, 0.9uM.
  • the present invention provides an induction medium for differentiation from (eg, human-derived) ectoderm to photoreceptor neuron cells, characterized in that LY2157299 and AM580 are added to NouvNeu-basal basal medium, and the NouvNeu-
  • basal basal medium is Duchenne's modified Eagle/F12 medium (DMEM/F12 medium), 0.1%-2% minimum essential medium non-essential amino acids (Minimum Essential Medium Non-Essential Amino Acids, the optimal concentration is 1%), 0.1-0.8g/L sodium chloride (Sodium Chloride, the optimum concentration is 0.5g/L), 0.1-80mg/L vitamin C (L-ascorbic acid, the optimum concentration is 64mg/L), 0.1 -1.8uM vitamin B12 (optimal concentration is 1.2uM), 0.1-9.8ng/ml progesterone (Progesterone, optimum concentration is 6.3ng/ml), 10-35ug/ml putrescine
  • the dosage of LY2157299 is 55nM-25uM, preferably 100nM, 300nM, 500nM, 700nM, 900nM, 1uM, 2.5uM, 5uM, 7.5uM, 10uM, 12.5uM, 15uM, 17.5uM, 20uM or 22uM.
  • the dosage of AM580 is 0.05-0.95uM, preferably 0.08uM, 0.09uM, 0.1uM, 0.2uM, 0.3uM, 0.5uM, 0.6uM, 0.7uM, 0.8uM, 0.9uM.
  • the dosage of the LY2157299 is 7.5uM, and the dosage of the AM580 is 0.5uM.
  • the present invention provides a method for differentiating pluripotent stem cells into photoreceptor neuron cells, wherein the pluripotent stem cells are added with 55nM-25uM LY2157299 (preferably 100nM, 300nM, 500nM, 700nM, 900nM, 1uM) , 2.5uM, 5uM, 7.5uM, 10uM, 12.5uM, 15uM, 17.5uM, 20uM or 22uM) in NouvNeu-basal basal medium to obtain ectodermal cells, and then the ectodermal cells were cultured in the above-mentioned photoreceptor neurons Culturing in a cell induction medium to obtain photoreceptor neuron cells, preferably, the culture is carried out in the presence of a basement membrane, more preferably, the basement membrane is Matrigel (STEMCELL Technologies), A combination of one or more of laminin and vitronectin, the formulation of the NouvNeu-basal basal medium is Dul
  • the present invention provides a photoreceptor neuron cell characterized by being cultured with the aforementioned induction medium for differentiation from (eg, human-derived) ectoderm into photoreceptor neuron cells.
  • the present invention provides a photoreceptor neuron cell, which is prepared by using the aforementioned method for differentiating (eg, human-derived) pluripotent stem cells into photoreceptor neuron cells.
  • the present invention provides a neural stem cell characterized by being cultured using the aforementioned induction medium for differentiation from (eg, human-derived) ectoderm into neural stem cells.
  • the present invention provides a neural stem cell characterized by being prepared by the aforementioned method for differentiating (eg, human-derived) ectoderm into neural stem cells.
  • the present invention provides an ectoderm cell characterized by being cultured using the aforementioned medium (eg, human-derived) pluripotent stem cells differentiated into (eg, human-derived) ectoderm.
  • medium eg, human-derived
  • pluripotent stem cells differentiated into eg, human-derived
  • the present invention provides an ectoderm cell characterized by being prepared by the aforementioned method for differentiating (eg, human-derived) pluripotent stem cells into (eg, human-derived) ectoderm.
  • the present invention relates to the use of the aforementioned photoreceptor neuron cells in the treatment of degenerative diseases of the optic nerve, such as age-related macular degeneration and pigmentary degeneration.
  • the present invention relates to the use of the aforementioned photoreceptor neuron cells in the transplantation of photoreceptor neurons.
  • the present invention relates to the use of ectodermal cells in the preparation of photoreceptor neuronal cells.
  • the present invention relates to the application of compound LY2157299 and/or JW55 in the development of nervous system in vitro, to the combination of the compound and basic medium, the culture of the combination in ectodermal cells or neural stem cells and its clinical application.
  • the present invention relates to the application of a compound (eg LY2157299, and/or AM580) in the in vitro development of the nervous system, the combination of the compound and a basal medium, the culture of the combination in photoreceptor nerve cells and its clinical application.
  • a compound eg LY2157299, and/or AM580
  • the present invention relates to a kit comprising NouvNeu-basal basal medium, LY2157299, and/or AM580, preferably, the amount of LY2157299 is 55nM-25uM 2.5uM, 5uM, 7.5uM, 10uM, 12.5uM, 15uM, 17.5uM, 20uM or 22uM, the amount of AM580 is 0.05-0.95uM, preferably 0.08uM, 0.09uM, 0.1uM, 0.2uM, 0.3uM, 0.5uM, 0.6uM, 0.7uM, 0.8uM, 0.9uM.
  • Monolayer adherent culture of pluripotent stem cells in serum-free ectoderm induction medium which contains chemical small molecules, amino acids, inorganic salts and other components used in the present invention, but does not contain serum, BMP or Substances and other components of the TGF signal transduction pathway were adhered and cultured using this serum-free neural induction medium for 15 days to obtain adherent neural stem cells.
  • the cells are further treated with vitamin pathway activators to obtain photoreceptor cells.
  • the substances that act on the BMP signal transduction pathway include one or more proteins that are freely arranged and combined from the following: BMP2, BMP4, BMP4, Smad1, Smad5, Smad8; wherein the act on Substances of the TGF signal transduction pathway include one or more proteins in a free arrangement of the following: Activin, TGF-beta, Nodal, Smad2, Smad3.
  • Vitamins include one or more of the following combinations of vitamins: vitamin C, vitamin E, vitamin B12, vitamin A.
  • the adherent culture is performed in the presence of a basement membrane preparation.
  • the basement membrane preparation of the present invention can form a thin film composed of extracellular matrix molecules on the surface of the culture vessel, and can provide support similar to the in vivo environment for parameters such as cell morphology, growth, differentiation and movement.
  • the basement membrane is a combination of one or more of Matrigel (STEMCELL Technologies), Laminin and Vitronectin.
  • the serum-free medium in the present invention means that it does not contain serum directly separated from blood.
  • Serum is the clear liquid portion of plasma that does not contain fibrinogen or blood cells and remains liquid after blood clots.
  • the serum-free medium may contain serum substitutes, and examples of serum substitutes include purified substances such as serum albumin, transferrin, fatty acids, and the like.
  • photoreceptor neuron cell In the present invention, the expressions "photoreceptor neuron cell”, “photoreceptor neuron cell” and “photoreceptor cell” are interchangeable.
  • Figure 1 shows: the ectodermal cells obtained using LY2157299, the differentiation of the obtained cells to neural stem cells and the results of molecular identification.
  • Figure 1a shows the neural stem cells obtained by the control method, and the structure of the neural rosette was reconstructed after passage in vitro;
  • Figure 1b shows the neural stem cells obtained by using LY2157299, and the structure of the neural rosette was reconstructed after passage in vitro;
  • Figure 1c shows the neural rosette obtained by the control method.
  • Stem cells after neural rosette reconstruction in vitro, can express neural stem cell markers Pax6 and Nestin;
  • Figure 1d shows that neural stem cells obtained using LY2157299, after neural rosette reconstruction in vitro, can express neural stem cell markers Pax6 and Nestin;
  • Figure 1e-1f Indicates the difference in the expression of neural stem cell markers in the process of neural stem cell formation compared with the contrast induction method (CK) using Q-PCR to compare the small molecule inhibitors of the present invention.
  • CK contrast induction method
  • Figure 2 shows: the use of different concentrations of LY2157299 for ectoderm cell induction, and the optimal concentration was screened.
  • Figure 2a shows the formation of ectodermal cell morphology using the LSB control;
  • Figure 2b shows that in the absence of LY2157299, cells do not form ectodermal morphology;
  • Figure 2c shows that low concentrations of LY2157299 produce mixed cell differentiation including ectodermal cells;
  • Figure 2d shows , an appropriate concentration of LY2157299 produced uniformly differentiated ectodermal cells;
  • Figure 2e shows that too high concentration of LY2157299 promotes the differentiation of ectodermal cells into neural cells;
  • Figure 2f shows that ectodermal markers are positively correlated with the concentration of LY2157299;
  • Figure 2g shows that apoptosis markers are associated with LY2157299 concentrations were positively correlated. Therefore, the optimal concentration of
  • Figure 3 shows that photoreceptor cells were induced using different concentrations of AM580, and the optimal concentration was screened.
  • Figure 3a shows the formation of ectodermal cell morphology using the LSB control;
  • Figure 3b shows that in the absence of AM580, cells form cortical neuron morphology with long axons;
  • Figure 3c shows that low concentrations of AM580 produce mixed cells including cortical neurons Differentiation;
  • Fig. 3d shows that an appropriate concentration of AM580 produces uniformly differentiated photoreceptor cell types with short axons;
  • Fig. 3e shows that a high concentration of AM580 induces synergism in differentiated cells;
  • Figure 4 shows: the induction of photoreceptor cells using different concentrations of AM580, the morphology of the obtained cells and the Q-PCR identification of photoreceptor markers.
  • Figures 4a-4d show photoreceptor cells formed using RA control and different concentrations of AM580;
  • Figure 4e shows the comparison of the expression of the neural cell marker Nestin in cells used in 4a-4d;
  • Figure 4f shows that the cells used in 4a-4d Comparison of the expression of the photoreceptor cell marker Rcoverin of the cells used;
  • Figure 4g shows the comparison of the expression of the photoreceptor cell marker Rhodopsin of the cells used in 4a-4d;
  • Figure 4h shows the expression of the cells used in 4a-4d.
  • Figure 4i shows the comparison of the expression of the photoreceptor cell marker Opsin1 (OPN1LW) for the cells used in 4a-4d.
  • OPN1LW photoreceptor cell
  • FIG. 5 Photoreceptor cells were induced with different concentrations of AM580, and the morphology of the obtained cells and the fluorescence immunological identification of photoreceptor markers. The results showed that the photoreceptor cells obtained from the RA control group expressed Nestin protein and Recoverin protein (5a), mature photoreceptor marker proteins CRX and Opsin protein (5b), Rhodopsin and PAX6 protein (5c). Consistent protein expression pattern in the RA control group (5d-5l).
  • FIG. 6 Schematic diagram of the concentration identification of key components in the basal culture system.
  • 6a Schematic diagram of the effect of different concentrations of sodium chloride on the osmotic pressure of the culture system
  • 6b Schematic diagram of the difference in expression of photoreceptor neurons obtained with different concentrations of vitamin B12
  • 6c The effect of insulin on the physiological state of cells during long-term cell culture Schematic diagram
  • 6d Schematic diagram of the effect of vitamin C on the physiological state of cells during long-term cell culture
  • 6e Schematic diagram of the effect of different concentrations of progesterone on the physiological state of cells during the induction of photoreceptor cells
  • 6f Schematic diagram of the effect of different concentrations of putrescine on photoreceptor cells Schematic diagram of the effect on the physiological state of cells during the induction of cells
  • 6g Schematic diagram of the effect of different concentrations of Optiferrin on the physiological state of the cells during the induction of photoreceptor cells.
  • the present invention establishes a serum-free and animal-derived substance-free photoreceptor neuron induction culture system. Therefore, photoreceptor neurons are obtained by reprogramming and directed differentiation of adult cells from various sources (such as human sources), and have complete biological functions, stable state between multiple batches, and high purity. , solves the problems of low purity and long cycle in the production process of cell-based drugs; and the invention also solves the long-standing problems of animal-derived culture methods and trophoblast contamination, and can be used for transplantation treatment of various visual disorders, so It has huge economic and social effects.
  • DMEM/F12 Medium Duchenne's Modified Eagle/F12 Medium (DMEM/F12 Medium, SIGMA), 0.1%-2% Minimum Essential Medium Non-Essential Amino Acids (Minimum Essential Medium Non-Essential Amino Acids, Cat. No.
  • 11140076 Thermo Fisher
  • 0.1 -0.8g/L sodium chloride sodium Chloride, 0.5g/L, SIGMA
  • 0.1-80mg/L vitamin C L-ascorbic acid, 64mg/L, SIGMA
  • 0.1-1.8uM vitamin B12 1.2uM, SIGMA
  • 0.1-9.8ng/ml progesterone Progesterone, 6.3ng/ml, SIGMA
  • 10-35ug/ml putrescine Putrescine, 23ug/ml, SIGMA), 1ug/L-25ug/L sodium selenite
  • the optimal concentration is 13.6ug/L, SIGMA), 0.1-44ug/ml insulin (22ug/ml, Solarbio), 0.5-20umol/L Optiferrin (10 ⁇ mol/L, SIGMA), 2.5-100ng/ml plant-derived recombinant human Basic growth factor (OsrbFGF, the optimal concentration is 50ng/m
  • the ectoderm cell induction medium of the present invention is prepared by adding 55nM-25uM LY2157299 (Selleck) (the optimum concentration is 7.5uM) (preferably 100nM, 300nM, 500nM, 700nM, 900nM, 1uM, 2.5uM, 5uM, 7.5uM, 10uM, 12.5uM, 15uM, 17.5uM, 20uM or 22uM).
  • LY induction method referred to as the LY induction method.
  • NouvNeu basal basal medium was supplemented with 100nM LDN-193189 (Selleck) and 10uM SB431542 (Selleck), hereinafter referred to as the LSB induction method.
  • Ectoderm cells are derived from human pluripotent stem cells, including embryonic pluripotent stem cells, such as the H9 cell line and human induced pluripotent stem cells. Among them, human induced pluripotent stem cells were obtained by reprogramming from CD34+ cells according to "Reprogramming medium and culture method for reprogramming induced pluripotent stem cells” (ZL201910050800.7). In the present invention, “induced pluripotent stem cells” and “pluripotent stem cells” are used interchangeably.
  • Human pluripotent stem cells were used to coat T25 cell culture flasks with Matrigel (STEMCELL Technologies), and then placed in a 37°C incubator for more than one hour after plating. 1x10 6 cells were seeded in T25 flasks for expansion and passage.
  • ectoderm cell induction use 50ug/ml polylysine (SIGMA, P6407) to coat a 6-well culture plate, place the plate in a 37°C incubator and incubate for more than 3 hours; then use 5ug/ml laminin (Laminin, I2020, SIGMA ALDRICH) was further coated and placed in a 37°C incubator to incubate for more than 3 hours after plating.
  • pluripotent stem cells reached 70% coverage, they were digested with EDTA at 37°C for 5 min, and DMEM was used to stop cell digestion. After the cells were washed and centrifuged, they were re-seeded in T25 culture plates at a ratio of 2 ⁇ 10 5 per flask.
  • the neural stem cell induction medium of the present invention is used for induction, and the medium is changed every day until lamellar ectoderm cells are formed.
  • Ectoderm was digested with EDTA for 5 min at 37°C, and cell digestion was terminated with DMEM. After the cells were washed and centrifuged, they were re-inoculated into T25 culture plates coated with 50ug/ml polylysine and 5ug/ml laminin according to the ratio of 2 ⁇ 10 5 per flask to carry out expansion culture.
  • the culture conditions were 37°C, 5% CO2 (Panasonic, MCO-18AC).
  • Ectoderm cells can further form neural stem cells.
  • use NouvNeu-basal basal medium in vitro add 7.5uM LY2157299 and 0.5-20uM JW55 (the optimal concentration is 5uM), and further cultivate examples The ectoderm cells obtained in 2.1, until the neural rosette structure is formed.
  • the LSB induction method was used as a control.
  • Neural stem cells obtained by LY-induced and LSB-induced ectoderm differentiation were used for identification by immunofluorescence staining: cells were fixed with 4% paraformaldehyde at room temperature for 40 minutes, washed twice with DPBS buffer; then with 0.1% Triton X -100 permeabilization for 5 min, washed twice with DPBS buffer; cells were then incubated overnight at 4°C in DPBS buffer containing 10% horse serum and 0.1% Triton X-100; antibody diluted in DPBS buffer was then added , incubated at 37°C for 2 hours, washed three times with DPBS buffer, and photographed with Leica DMi8. Antibody usage details are shown in Table 1. The results are shown in Figures 1a-1d, showing that both the LY induction method and the control LSB induction method can induce the generation of neural stem cells with neural rosette structures, and the obtained neural stem cells express Pax6 and Nestin.
  • Transcriptional changes of different marker genes during induction from ectodermal cells to neural stem cells were detected using Q-PCR.
  • the ectoderm cells used in Example 2 the neural stem cells obtained by the LY induction method and the LSB induction control method, were used for total RNA extraction using RNeasy Mini or Micro Kit (QIAGEN) respectively, and 1 mg RNA was used SuperScript III First-Strand Synthesis System (Invitrogen) to synthesize cDNA.
  • SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa) were used for Quantitative PCR labeling and reaction, and beta-Actin was used as an internal reference. All data were analyzed with the delta-Ct method.
  • the primer sequences used to identify the coding genes of different cellular markers are shown in Table 2.
  • the results are shown in Figures 1e-1g.
  • the results showed that the neural stem cells induced by the LY-induced method and the control LSB-induced method expressed a variety of neural stem cell markers SOX2, PAX6 and Nestin at the transcriptional level. Therefore, the ectoderm obtained by the present invention has the ability to further differentiate into neural stem cells.
  • LY2157299 The effects of different concentrations of LY2157299 on the formation of epidermal cells were detected by Q-PCR.
  • the induced pluripotent stem cells used in Example 2 using LY2157299 at different concentrations in the LY induction method and the ectoderm cells obtained by the LSB induction method, were used for total RNA extraction using RNeasy Mini or Micro Kit (QIAGEN), and 1 mg RNA was extracted with SuperScript III cDNA was synthesized by First-Strand Synthesis System (Invitrogen). SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa) were used for Quantitative PCR labeling and reaction, and beta-Actin was used as an internal reference.
  • SOX2-F CATGCAGGTTGACACCGTTGG (SEQ ID NO: 1) SOX2-R ATGGATTCTCGGCAGACTGATTCA (SEQ ID NO: 2) PAX6-F TCTTTGCTTGGGAAATCCG (SEQ ID NO: 3) PAX6-R CTGCCCGTTCAACATCCTTAG (SEQ ID NO: 4) Nestin-F TCAAGATGTCCCTCAGCCTGGA (SEQ ID NO: 9) Nestin-R TGGCACAGGTGTCTCAAGGGTAG (SEQ ID NO: 10) CASP3-F GGAAGCGAATCAATGGACTCTGG (SEQ ID NO: 5)
  • the ectodermal cells obtained by the NouvNeu system in Example 2 were subjected to directed differentiation in NouvNeu medium.
  • 50ug/ml polylysine (SIGMA, P6407) was used to coat a 6-well culture plate, and after plating, it was placed in a 37°C incubator and incubated for more than 3 hours until cells were seeded.
  • the photoreceptor neuron cell induction medium of the present invention is prepared by adding 55nM-25uM LY2157299 (the optimum concentration is 7.5uM) (preferably 100nM, 300nM, 500nM, 700nM, 900nM, 1uM, 2.5uM) in the NouvNeu-basal basal medium uM, 5uM, 7.5uM, 10uM, 12.5uM, 15uM, 17.5uM, 20uM or 22uM) and 0.05-0.95uM AM580 (Selleck) (optimal concentration is 0.5uM) (preferably 0.08uM, 0.09uM, 0.1uM, 0.2uM, 0.3uM, 0.5uM, 0.6uM, 0.7uM, 0.8uM, 0.9uM). until the cells form photoreceptor neuron cells. Add 0.5uM retinoic acid to NouvNeu medium to form a control experiment, hereinafter referred to as RA.
  • RA 0.5uM
  • the AM580 concentration screening test was carried out with reference to Example 2.3, and the expression differences of photoreceptor neurons obtained by different concentrations of AM580 in the AM580 induction method and the LSB induction method were compared.
  • the list of primers used is shown in Table 2 and Table 4.
  • the results showed that AM580 concentration was positively correlated with the photoreceptor non-specific marker PAX6, but when AM580 concentration was too high, apoptosis was induced.
  • the concentration of AM580 used in the present invention is the optimal concentration.
  • the culture conditions were 37°C, 5% CO2 (Panasonic, MCO-18AC).
  • the two control materials in each group were taken for immunofluorescence staining identification: cells were fixed with 4% paraformaldehyde at room temperature for 40 minutes, washed twice with DPBS buffer, and then permeabilized with 0.1% Triton X-100 for 5 min, washed twice with DPBS buffer; cells were then incubated overnight at 4°C with 10% horse serum and 0.1% Triton X-100 in DPBS buffer; then 2% horse serum and 0.1% Triton X-100 in DPBS were added The primary antibody diluted in buffer was incubated at 37°C for 2 hours, washed three times with DPBS buffer, and the secondary antibody diluted in 2% horse serum and 0.1% Triton X-100 in DPBS buffer was added to take pictures.
  • Transcriptional changes of different marker genes during induction from ectodermal cells to photoreceptor neurons were detected using Q-PCR.
  • SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa) were used for Quantitative PCR labeling and reaction, and beta-Actin was used as an internal reference.
  • CRX-F CTTGTAGAGGACGCAGTCTCCATCT (SEQ ID NO: 11) CRX-R CAATCGTGCCAGACGGTGTTAGG (SEQ ID NO: 12) OPN1LW-F TCATCGCCAGCACTATCAGCATTG (SEQ ID NO: 13) OPN1LW-R ACAGCAGACCAGATCCAGGAGAAG (SEQ ID NO: 14) Recoverin-F CAGTTCCAGAGCATCTACGCCAAG (SEQ ID NO: 15) Recoverin-R GCCATGACGATCTCCAGCACTTC (SEQ ID NO: 16) Rhodopsin-F CAGCGTGGCATTCTACATCTTCAC (SEQ ID NO: 17)
  • Rhodopsin-R ACCTGGCTCGTCTCCGTCTT (SEQ ID NO: 18) PAX6-F TCTTTGCTTGGGAAATCCG (SEQ ID NO: 19) PAX6-R CTGCCCGTTCAACATCCTTAG (SEQ ID NO: 20) ⁇ Actin-F GGCCGAGGACTTTGATTGCACA (SEQ ID NO: 21) ⁇ Actin-R GGGCACGAAGGCTCATCATTCAA (SEQ ID NO: 22)
  • the formula of NouvNeu-basal basal medium shown in the examples of the present invention is Duchenne's modified Eagle/F12 medium (DMEM/F12 medium), 0.1%-2% minimum essential medium non-essential amino acids (Minimum Essential Medium Non-Essential Amino Acids, the optimal concentration is 1%), 0.1-0.8g/L Sodium Chloride (Sodium Chloride, the optimal concentration is 0.5g/L), 0.1-80mg/L Vitamin C (L-ascorbic acid, The optimum concentration is 64mg/L), 0.1-1.8uM vitamin B12 (the optimum concentration is 1.2uM), 0.1-9.8ng/ml progesterone (Progesterone, the optimum concentration is 6.3ng/ml), 10-35ug/ml Amine (Putrescine, the optimum concentration is 23ug/ml), 1ug/L-25ug/L sodium selenite (the optimum concentration is 13.6ug/L), 0.1-44ug/ml insulin (
  • the vitamin B12 concentration screening test was carried out with reference to Example 2.3, and the expression differences of photoreceptor neuron cells obtained by using different concentrations of vitamin B12 were compared.
  • the list of primers used is shown in Table 2 and Table 4.
  • Figure 6b the results showed that vitamin B12 concentration was positively correlated with the photoreceptor non-specific marker PAX6, but when the vitamin B12 concentration was too high, it caused non-specific differentiation of cells, resulting in a decrease in the relative expression of PAX6. Therefore, the concentration of vitamin B12 used in the present invention is the optimal concentration for use.
  • the Cyquant test was also used to detect the cell viability of progesterone, putrescine and Optiferrin, so as to study the effect of different concentrations of progesterone, putrescine and Optiferrin on the physiological state of cells during the induction of photoreceptor cells, the results are shown in Figure 6e, 6f , and as shown in Figure 6g, the concentrations of progesterone, putrescine and Optiferrin were all positively correlated with the cell proliferation rate. Combined with the cost factor, the range of progesterone, putrescine and Optiferrin used in the present invention belonged to the best use range.

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Abstract

提供了一种光感受器神经元细胞纯化学诱导方法，通过将一组小分子抑制剂添加至无血清基础培养基中，将多能干细胞快速转化为光感受器神经元细胞，本发明不使用血清，使用化学小分子替代视黄醇，保证了光感受器神经元发育所需要的视觉循环的运行的同时也排除了血清的不利因素。

Description

一种光感受器神经元细胞的化学诱导方法 技术领域
本发明属于生物领域。具体涉及光感受器神经元细胞的化学诱导及培养方法、培养基及其在多种疾病中的应用。
背景技术
外胚层是胚胎发育过程中形成的最外层，随着器官发生的开始，外胚层细胞逐渐分化为大脑、脊髓、感觉器官等重要的系统。其中神经系统是负责思维、情感、感知、运动等功能的重要系统。相比于肿瘤等疾病，目前神经系统疾病的药物数量较少，且研发周期漫长，其中最为重要的一个原因是外胚层谱系中多种原代细胞的特殊性，例如原代神经元的不可再生性导致了神经系统药物体外药物筛选平台的稀缺。外胚层细胞的体外再生能解决多种退行性疾病，例如神经退行性疾病是目前常见的老龄化疾病，治疗及护理费用极其昂贵，而且市面上无特效药物可以进行有效的治疗。神经退行性疾病包括肌萎缩侧索硬化症(ALS)、帕金森病(PD)、阿尔茨海默症(AD)等疾病。世界卫生组织统计，2050年我国神经退行性疾病患者将超过3000万，预期医疗费用将超过1万亿。此类疾病的治疗难点在于中枢神经系统的神经细胞具有不可再生性，而这类疾病是由于中枢神经的不可逆损伤造成。目前，神经细胞的移植是最为有效的一种治疗方式。
视觉神经的退行性疾病也是其中的一大类疾病。以与年龄相关的黄斑变性(AMD)为例，它是50岁以上人群视力受损的主要原因，其特征是视网膜退化，可导致中央视力丧失。此外，色素变性这种罕见遗传性疾病，其特征是视网膜中的光感受器逐渐退化，无法将光信号转换成向大脑的电信号。视网膜是高等动物体中能够感受“影像”的重要组织，由光敏细胞和光感受器组成。光感受器能将影像的光信号转变为神经信号，是能让大脑“看见”影像的重要细胞。这其中，锥形受体细胞即视锥细胞负责我们的日间视觉及颜色的感知，视杆细胞，则更为敏感，即使在低光照条件也可以感知到微弱的光。感光细胞的受损，是视网膜退行性疾病的重要成因，导致眼球无法将“影像”转化的神经信号传递到大脑，从而严重影响人的视力，甚至导致失明。更为严峻的是，光感受器神经细胞的受损是一种不可逆的过程，无法自我修复，因此此类疾病没有良好的治疗方法。部分患者采取视网膜移植方法，但视网膜资源有限，多来自于遗体捐赠，远不能满足临床需要；人工视网膜的原理是替换这些光感受器。该装置由固定在视网膜和电极化合物下的植入物组成，这些植入物可刺激视网膜神经元向大脑传递信息，但目前人工视网膜价格昂贵，且不能精细恢复视觉功能；除此之外，还有基于病毒载体的基因疗法，通过修正遗传突变让遗传性光感受器恢复功能，同样，基因疗法除了价格昂贵之外，并不能解决由年龄造成的视觉神经的退行性疾病。
2006年，山中伸弥的团队发明了一种由OCT4、SOX2、KLF4和c-Myc四种转录因子构成的“鸡尾酒”法，能够成功将终端分化的皮肤成纤维细胞重编程成为具有分化多能性的干细胞，这种干细胞被称为诱导多能干细胞(Induced Pluripotent Cells)(Cell，2006，124(4)pp.663-676；Cell，2007，131(5)pp.861-872)。这些干细胞具有和胚胎干细胞(Embryonic Stem Cells)类似的分化潜能，能够形成人体发育最基本的三个胚层：外胚层、中胚层及内胚层，并最终形成多种成体细胞。这一发明突破了在医学上使用人胚胎干细胞的伦理限制，可以解决细胞移植治疗中的免疫排斥问题，大大拓展了干细胞技术在临床医学上的应用潜力。 使用包括胚胎干细胞及iPSC在内的全能干细胞或者多能干细胞作为原材料进行外胚层细胞诱导分化可以作为临床治疗的新思路，大大拓展了外胚层细胞在临床医学上的应用潜力。
以神经系统为例，目前在再生医学领域，神经干细胞及神经元的诱导多采取SMAD途径双重抑制法(Dual SMAD inhibition)(Nat Biotechnol，2009，05；27(5)：485)，该方法得到的神经干细胞能够分化为其它类型的神经元细胞，其原理是通过抑制BMP和TGFBeta途径来模拟胚胎发育早期的信号途径，从而诱导神经干细胞的产生。其中LDN-193189和SB431542是广为使用的两种化学小分子抑制剂，分别通过作用于BMP4途径中的ALK2和ALK3，以及TGFBeta途径中的ALK5来实现移植内胚层及中胚层形成，从而起到神经发生的作用。通过这种方法，可以在血清类成分(Knockout Serum Replacement)胎牛血清(FBS)和视黄酸的存在下得到光感受器神经元，且生产周期至少需要70天以上(STEM CELLS TRANSLATIONAL MEDICINE，2018；7:210-219；Sci Transl Med，2019.1 16；11(475))。但是血清提取物，动物源性提取物的存在，对细胞治疗领域，特别是细胞制药来说，是一种不利于药物安全性的潜在危险，例如动物源性病毒的混入，以及致敏性蛋白的存在，都可能会导致治疗效果的失败。此外，视黄酸是视觉循环的重要参与组成成分，涉及通过视网膜色素上皮(RPE)将维生素A(称为全反式视黄醇或全反式ROL)转化为11-顺式视黄醛(11-顺式-RAL)，转运11-顺式-RAL转化为感光体，产生光色素，将11-顺式-RAL异构化并还原成全反式ROL，并将全反式ROL转运回RPE参与另一个循环(Nature.1960；168:114-118；Nat.Genet.1997；15:236-246；J.Biol.Chem.1997；272:10303-10310)。细胞培养体系中的类维生素A，例如视黄酸(RA)，非常容易受到氧化而失去活性。因此，为了稳定视黄酸不被快速氧化损伤，血清被大量用来稳定细胞培养体系中的视黄酸(Int J Dev Biol.2012；56(4):273-8)。因此，一种既不依赖于血清，又能在细胞培养条件下稳定存在的类维生素A化学分子，对光感受器神经元的体外稳定安全诱导极为重要。
本发明使用的Galunisertib(LY2157299)，标准化学命名：4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6-carboxamide或4-(2-(6-甲基吡啶-2-基)-5,6-二氢-4H-吡咯并[1,2-b]吡唑-3-基)喹啉-6-甲酰胺；为TGF-β受体Ⅰ(TGF-βRI,ALK5)小分子抑制剂。LY2157299目前正处于II期临床评估，针对肝癌和胶质母细胞瘤的抗癌活性(Giannelli G1,Villa E,Lahn M.Transforming Growth Factor-βas a Therapeutic Target in Hepatocellular Carcinoma.Cancer Res.2014.4.1；74(7):1890-4)，通过阻断TGF-β信号通路抑制肿瘤的生长、侵袭和转移过程；此外，多项研究表明LY2157299阻断了CTGF的产生并抑制了新血管生成，从而抑制了癌细胞的生长。目前，该分子的发展方向主要是基于肺癌、肝癌和胶质母细胞瘤的药物开发以及治疗(Pharmaceutics.2020.5.18；12(5):459)，并没有公开任何在神经再生领域的应用。
本发明使用了一种无血清小分子化学诱导体系，将多能干细胞快速转化为光感受器神经细胞，与目前国际通行的血清加视黄醇的诱导方法相比，本发明不使用血清，使用化学小分子替代视黄醇，保证了光感受器神经元发育所需要的视觉循环的运行的同时也排除了血清的不利因素。因此，本发明不仅完全免除了在细胞培养过程中动物源性成分带来的潜在危险，而且全新的化学小分子的组合展现出极大的纯度及产率优势。所以，本发明大大拓展了光感受器神经细胞移植的临床前景。同时解决了同类细胞产品生产过程中纯度低，周期长的问题，因此具有巨大的经济效应和社会效应。
发明内容
本发明涉及光感受器神经元细胞的化学诱导及培养方法、培养基及其在多种疾病中的应用。
具体地，本发明涉及如下几个方面：
一方面，本发明提供LY2157299在(例如人源)多能干细胞分化为外胚层中的应用。
另一方面，本发明提供了一种从(例如人源)多能干细胞分化为(例如人源)外胚层的培养基。
进一步地，所述从(例如人源)多能干细胞分化为(例如人源)外胚层的培养基，其特征在于，在NouvNeu-basal基础培养基中加入LY2157299，所述NouvNeu-basal基础培养基的配方为杜氏改良伊格尔/F12培养基(DMEM/F12培养基)、0.1％-2％最低必须培养基非必需氨基酸(Minimum Essential Medium Non-Essential Amino Acids，最佳浓度为1％)、0.1-0.8g/L氯化钠(Sodium Chloride，最佳浓度为0.5g/L)、0.1-80mg/L维生素C(L-ascorbic acid，最佳浓度为64mg/L)、0.1-1.8uM维生素B12(最佳浓度为1.2uM)、0.1-9.8ng/ml黄体酮(Progesterone，最佳浓度6.3ng/ml)、10-35ug/ml腐胺(Putrescine，最佳浓度为23ug/ml)、1ug/L-25ug/L亚硒酸钠(最佳浓度为13.6ug/L)、0.1-44ug/ml胰岛素(最佳浓度为22ug/ml)、0.5-20umol/L Optiferrin(最佳浓度为10μmol/L)、2.5-100ng/ml植物源重组人碱性生长因子(OsrbFGF，最佳浓度为50ng/mL)。
进一步地，LY2157299的用量为55nM-25uM，优选为100nM、300nM、500nM、700nM、900nM、1uM、2.5uM、5uM、7.5uM、10uM、12.5uM、15uM、17.5uM、20uM或22uM。
进一步地，所述LY2157299的用量为7.5uM。
另一方面，本发明提供一种(例如人源)多能干细胞分化为(例如人源)外胚层的方法，其特征在于，将多能干细胞在添加55nM-25uM LY2157299(优选为100nM、300nM、500nM、700nM、900nM、1uM、2.5uM、5uM、7.5uM、10uM、12.5uM、15uM、17.5uM、20uM或22uM)的NouvNeu-basal基础培养基中培养，获得外胚层细胞，所述NouvNeu-basal基础培养基的配方为杜氏改良伊格尔/F12培养基(DMEM/F12培养基)、0.1％-2％最低必须培养基非必需氨基酸(Minimum Essential Medium Non-Essential Amino Acids，最佳浓度为1％)、0.1-0.8g/L氯化钠(Sodium Chloride，最佳浓度为0.5g/L)、0.1-80mg/L维生素C(L-ascorbic acid，最佳浓度为64mg/L)、0.1-1.8uM维生素B12(最佳浓度为1.2uM)、0.1-9.8ng/ml黄体酮(Progesterone，最佳浓度6.3ng/ml)、10-35ug/ml腐胺(Putrescine，最佳浓度为23ug/ml)、1ug/L-25ug/L亚硒酸钠(最佳浓度为13.6ug/L)、0.1-44ug/ml胰岛素(最佳浓度为22ug/ml)、0.5-20umol/L Optiferrin(最佳浓度为10μmol/L)、2.5-100ng/ml植物源重组人碱性生长因子(OsrbFGF，最佳浓度为50ng/mL)。
另一方面，本发明提供LY2157299和JW55在(例如人源)多能干细胞分化为神经干细胞中的应用。
另一方面，本发明提供将(例如人源)多能干细胞分化为神经干细胞的培养基，其特征在于，在NouvNeu-basal基础培养基中加入LY2157299和JW55，所述NouvNeu-basal基础培养基的配方为杜氏改良伊格尔/F12培养基(DMEM/F12培养基)、0.1％-2％最低必须培养基非必需氨基酸(Minimum Essential Medium Non-Essential Amino Acids，最佳浓度为1％)、0.1-0.8g/L氯化钠(Sodium Chloride，最佳浓度为0.5g/L)、0.1-80mg/L维生素C(L-ascorbic acid，最佳浓度为64mg/L)、0.1-1.8uM维生素B12(最佳浓度为1.2uM)、0.1-9.8ng/ml黄体酮(Progesterone，最佳浓度6.3ng/ml)、10-35ug/ml腐胺(Putrescine，最佳浓度为23ug/ml)、 1ug/L-25ug/L亚硒酸钠(最佳浓度为13.6ug/L)、0.1-44ug/ml胰岛素(最佳浓度为22ug/ml)、0.5-20umol/L Optiferrin(最佳浓度为10μmol/L)、2.5-100ng/ml植物源重组人碱性生长因子(OsrbFGF，最佳浓度为50ng/mL)；任选地，所述JW55的浓度为0.5-20uM，最佳浓度为5uM；任选地，所述LY2157299的用量为55nM-25uM，优选为100nM、300nM、500nM、700nM、900nM、1uM、2.5uM、5uM、7.5uM、10uM、12.5uM、15uM、17.5uM、20uM或22uM。
另一方面，本发明提供将(例如人源)外胚层分化为神经干细胞的培养基，其特征在于，在NouvNeu-basal基础培养基中加入LY2157299和JW55，所述NouvNeu-basal基础培养基的配方为杜氏改良伊格尔/F12培养基(DMEM/F12培养基)、0.1％-2％最低必须培养基非必需氨基酸(Minimum Essential Medium Non-Essential Amino Acids，最佳浓度为1％)、0.1-0.8g/L氯化钠(Sodium Chloride，最佳浓度为0.5g/L)、0.1-80mg/L维生素C(L-ascorbic acid，最佳浓度为64mg/L)、0.1-1.8uM维生素B12(最佳浓度为1.2uM)、0.1-9.8ng/ml黄体酮(Progesterone，最佳浓度6.3ng/ml)、10-35ug/ml腐胺(Putrescine，最佳浓度为23ug/ml)、1ug/L-25ug/L亚硒酸钠(最佳浓度为13.6ug/L)、0.1-44ug/ml胰岛素(最佳浓度为22ug/ml)、0.5-20umol/L Optiferrin(最佳浓度为10μmol/L)、2.5-100ng/ml植物源重组人碱性生长因子(OsrbFGF，最佳浓度为50ng/mL)；任选地，所述JW55的浓度为0.5-20uM JW55，最佳浓度为5uM；任选地，所述LY2157299的用量为55nM-25uM，优选为100nM、300nM、500nM、700nM、900nM、1uM、2.5uM、5uM、7.5uM、10uM、12.5uM、15uM、17.5uM、20uM或22uM。
另一方面，本发明提供(例如人源)多能干细胞分化为神经干细胞的方法，其特征在于，利用前述将(例如人源)多能干细胞分化为神经干细胞的培养基培养。
另一方面，本发明提供(例如人源)外胚层细胞分化为神经干细胞的方法，其特征在于，利用前述将(例如人源)外胚层分化为神经干细胞的培养基培养。
另一方面，本发明提供LY2157299和AM580在(例如人源)多能干细胞分化为光感受器神经元细胞中的应用。
进一步地，LY2157299的用量为55nM-25uM，优选为100nM、300nM、500nM、700nM、900nM、1uM、2.5uM、5uM、7.5uM、10uM、12.5uM、15uM、17.5uM、20uM或22uM。
进一步地，AM580的用量为0.05-0.95uM，优选为0.08uM，0.09uM，0.1uM，0.2uM，0.3uM，0.5uM，0.6uM，0.7uM，0.8uM，0.9uM。
另一方面，本发明提供一种从(例如人源)外胚层分化到光感受器神经元细胞的诱导培养基，其特征在于：在NouvNeu-basal基础培养基中加入LY2157299以及AM580，所述NouvNeu-basal基础培养基的配方为杜氏改良伊格尔/F12培养基(DMEM/F12培养基)、0.1％-2％最低必须培养基非必需氨基酸(Minimum Essential Medium Non-Essential Amino Acids，最佳浓度为1％)、0.1-0.8g/L氯化钠(Sodium Chloride，最佳浓度为0.5g/L)、0.1-80mg/L维生素C(L-ascorbic acid，最佳浓度为64mg/L)、0.1-1.8uM维生素B12(最佳浓度为1.2uM)、0.1-9.8ng/ml黄体酮(Progesterone，最佳浓度6.3ng/ml)、10-35ug/ml腐胺(Putrescine，最佳浓度为23ug/ml)、1ug/L-25ug/L亚硒酸钠(最佳浓度为13.6ug/L)、0.1-44ug/ml胰岛素(最佳浓度为22ug/ml)、0.5-20umol/L Optiferrin(最佳浓度为10μmol/L)、2.5-100ng/ml植物源重组人碱性生长因子(OsrbFGF，最佳浓度为50ng/mL)。
进一步地，所述LY2157299的用量为55nM-25uM，优选为100nM、300nM、500nM、700nM、900nM、1uM、2.5uM、5uM、7.5uM、10uM、12.5uM、15uM、17.5uM、20uM或22uM。
进一步地，所述AM580的用量为0.05-0.95uM，优选为0.08uM，0.09uM，0.1uM，0.2uM，0.3uM，0.5uM，0.6uM，0.7uM，0.8uM，0.9uM。
进一步地，所述LY2157299的用量为7.5uM，所述AM580的用量为0.5uM。
另一方面，本发明提供一种多能干细胞分化为光感受器神经元细胞的方法，其特征在于，将多能干细胞在添加55nM-25uM LY2157299(优选为100nM、300nM、500nM、700nM、900nM、1uM、2.5uM、5uM、7.5uM、10uM、12.5uM、15uM、17.5uM、20uM或22uM)的NouvNeu-basal基础培养基中培养，获得外胚层细胞，然后将外胚层细胞在上述的光感受器神经元细胞诱导培养基中培养，获得光感受器神经元细胞，优选地，所述培养是在有基底膜的情况下进行的，更优选地，所述基底膜为基底胶(Matrigel)(STEMCELL Technologies)，层粘蛋白(Laminin)和玻连蛋白的一种或多种的组合，所述NouvNeu-basal基础培养基的配方为杜氏改良伊格尔/F12培养基(DMEM/F12培养基)、0.1％-2％最低必须培养基非必需氨基酸(Minimum Essential Medium Non-Essential Amino Acids，最佳浓度为1％)、0.1-0.8g/L氯化钠(Sodium Chloride，最佳浓度为0.5g/L)、0.1-80mg/L维生素C(L-ascorbic acid，最佳浓度为64mg/L)、0.1-1.8uM维生素B12(最佳浓度为1.2uM)、0.1-9.8ng/ml黄体酮(Progesterone，最佳浓度6.3ng/ml)、10-35ug/ml腐胺(Putrescine，最佳浓度为23ug/ml)、1ug/L-25ug/L亚硒酸钠(最佳浓度为13.6ug/L)、0.1-44ug/ml胰岛素(最佳浓度为22ug/ml)、0.5-20umol/L Optiferrin(最佳浓度为10μmol/L)、2.5-100ng/ml植物源重组人碱性生长因子(OsrbFGF，最佳浓度为50ng/mL)。
另一方面，本发明提供一种光感受器神经元细胞，其特征在于利用前面所述的从(例如人源)外胚层分化到光感受器神经元细胞的诱导培养基培养。
另一方面，本发明提供一种光感受器神经元细胞，其特征在于利用前面所述的(例如人源)多能干细胞分化为光感受器神经元细胞的方法制备。
另一方面，本发明提供一种神经干细胞，其特征在于利用前面所述的从(例如人源)外胚层分化到神经干细胞的诱导培养基培养。
另一方面，本发明提供一种神经干细胞，其特征在于利用前面所述的(例如人源)外胚层分化为神经干细胞的方法制备。
另一方面，本发明提供一种外胚层细胞，其特征在于利用前面所述的(例如人源)多能干细胞分化为(例如人源)外胚层的培养基培养。
另一方面，本发明提供一种外胚层细胞，其特征在于利用前面所述的(例如人源)多能干细胞分化为(例如人源)外胚层的方法制备。
另一方面，本发明涉及前面所述的光感受器神经元细胞在视觉神经的退行性疾病的治疗中的用途，所述视觉神经的退行性疾病是例如与年龄相关的黄斑变性、色素变性。
另一方面，本发明涉及前面所述的光感受器神经元细胞在光感受器神经细胞移植中的用途。
另一方面，本发明涉及外胚层细胞在制备光感受器神经元细胞中的用途。
本发明涉及化合物LY2157299和/或JW55在神经系统体外发育中的应用，涉及到该化合物与基础培养基的组合，以及这一组合在外胚层细胞或神经干细胞中的培养及其临床应用。
本发明涉及化合物(例如LY2157299，和/或AM580)在神经系统体外发育中的应用，涉及到该化合物与基础培养基的组合，以及这一组合在光感受器神经细胞中的培养及其临床应用。
本发明涉及一种试剂盒，其包含NouvNeu-basal基础培养基，LY2157299，和/或AM580， 优选地，所述LY2157299的用量为55nM-25uM优选为100nM、300nM、500nM、700nM、900nM、1uM、2.5uM、5uM、7.5uM、10uM、12.5uM、15uM、17.5uM、20uM或22uM，所述AM580的用量为0.05-0.95uM，优选为0.08uM，0.09uM，0.1uM，0.2uM，0.3uM，0.5uM，0.6uM，0.7uM，0.8uM，0.9uM。
本发明人诱导光感受器神经元细胞制备步骤：
在无血清外胚层诱导培养基中对多能干细胞进行单层贴壁培养，所述无血清培养基中含有本发明使用的化学小分子、氨基酸、无机盐等成分，而不含有血清、BMP或者TGF信号转导途径的物质等成分，使用这一无血清神经诱导培养基贴壁培养15天，得到贴壁神经干细胞。进一步使用维生素途径激活剂处理细胞，从而获得光感受器细胞。
在具体的实施方案中，所述作用于BMP信号转导途径的物质包括一个或者多个由下列各项自由排列组合的蛋白质：BMP2，BMP4，BMP4，Smad1，Smad5，Smad8；其中所述作用于TGF信号转导途径的物质包括一个或者多个由下列各项自由排列组合的蛋白质：Activin，TGF-beta，Nodal，Smad2，Smad3。维生素包括一个或者多个由下列各项排列组合的维生素：维生素C，维生素E，维生素B12，维生素A。
在具体的实施方案中，所述贴壁培养是在有基底膜制剂的情况下进行的。本发明所述基底膜制剂，可以在培养器皿的表面形成一层由胞外基质分子构成的薄膜，可以为细胞的形态、生长分化以及运动等参数提供与体内环境类似的支持。在具体的实施方案中，所述基底膜为基底胶(Matrigel)(STEMCELL Technologies)，层粘蛋白(Laminin)和玻连蛋白的一种或多种的组合。
本发明中的无血清培养基表示不含有直接从血液中分离的血清。血清为血浆的透明液体部分，其不含纤维蛋白原或血细胞，并且在血液凝固后保持为液体。无血清培养基可以含有血清替代物，血清替代物的实例包括血清白蛋白，转铁蛋白，脂肪酸等纯化物质。
在本发明中，表述“光感受器神经元细胞”、“光感受器神经细胞”和“光感受器细胞”可以互换。
附图说明
1.图1示出:使用LY2157299获得的外胚层细胞，所获细胞向神经干细胞分化及其分子鉴定结果。图1a表示使用对照方法获得的神经干细胞，经过体外传代后重建神经花环的结构；图1b表示使用LY2157299获得的神经干细胞，经过体外传代后重建神经花环的结构；图1c表示使用对照方法获得的神经干细胞，经过体外重建神经花环，能够表达神经干细胞的标志物Pax6和Nestin；图1d表示使用LY2157299获得的神经干细胞，经过体外重建神经花环，能够表达神经干细胞的标志物Pax6和Nestin；图1e-1f表示使用Q-PCR比较本发明的小分子抑制剂，与对比诱导方法(CK)相比在神经干细胞形成过程中神经干细胞标志物的表达差异。以上结果显示，LY2157299不仅能够形成神经干细胞，且与对照方法相比，在神经干细胞标志物的表达上比对照方法略有增加。
2.图2示出：使用不同浓度LY2157299进行外胚层细胞诱导，对最佳浓度进行筛选。图2a显示，使用LSB对照形成外胚层细胞形态；图2b显示，没有LY2157299时，细胞不形成外胚层形态；图2c显示，低浓度LY2157299产生包括外胚层细胞在内的混合细胞分化；图2d显示，适当浓度LY2157299产生均匀分化的外胚层细胞；图2e显示，过高浓度LY2157299促进外胚层细胞向神经细胞分化；图2f显示外胚层标志物与LY2157299浓度呈正相关；图2g显示凋亡标志物与LY2157299浓度呈正相关。因此，本发明使用的LY2157299最佳浓度为7.5uM。
3.图3示出：使用不同浓度AM580进行光感受器细胞诱导，对最佳浓度进行筛选。图3a显示，使用LSB对照形成外胚层细胞形态；图3b显示，没有AM580时，细胞形成具有长轴突的皮质神经元形态；图3c显示，低浓度AM580产生包括皮质神经元在内的混合细胞分化；图3d显示，适当浓度AM580产生均匀分化的具有短轴突的光感受器细胞类型；图3e显示，过高浓度AM580使分化细胞产生协迫；图3f显示外胚层及光感受器标志物PAX6与AM580浓度呈正相关；图3g显示凋亡标志物与AM580浓度呈正相关。因此，本发明中AM580最佳浓度为0.5uM。
4.图4示出：使用不同浓度AM580进行光感受器细胞诱导，所获细胞的形态及光感受器标志物的Q-PCR鉴定。图4a-4d显示，使用RA对照和不同浓度AM580形成的光感受器细胞；图4e显示，4a-4d中所使用的细胞的神经细胞标志物Nestin的表达比较；图4f显示，4a-4d中所使用的细胞的光感受器细胞标志物Rcoverin的表达比较；图4g显示，4a-4d中所使用的细胞的光感受器细胞标志物Rhodopsin的表达比较；图4h显示，4a-4d中所使用的细胞的光感受器细胞标志物CRX的表达比较；图4i显示4a-4d中所使用的细胞的光感受器细胞标志物Opsin1(OPN1LW)的表达比较。
图5：使用不同浓度AM580进行光感受器细胞诱导，所获细胞的形态及光受体标志物的荧光免疫鉴定。结果表明，RA对照组获得的光感受器细胞表达Nestin蛋白和Recoverin蛋白(5a)、成熟光感受器的标志蛋白CRX和Opsin蛋白(5b)、Rhodopsin和PAX6蛋白(5c)；AM580不同浓度组也表达和RA对照组一致的蛋白表达模式(5d-5l)。
图6：基础培养体系中关键成分的浓度鉴定示意图。6a：不同浓度氯化钠对培养体系渗透压的影响示意图；6b：使用不同浓度维生素B12得到的光感受器神经元细胞的表达差异示意图；6c：胰岛素在细胞长期培养过程中对细胞生理状态的影响示意图；6d：维生素C在细胞长期培养过程中对细胞生理状态的影响示意图；6e：不同浓度黄体酮在光感受器细胞诱导过程中对细胞生理状态的影响示意图；6f：不同浓度腐胺在光感受器细胞诱导过程中对细胞生理状态的影响示意图；6g：不同浓度Optiferrin在光感受器细胞诱导过程中对细胞生理状态的影响示意图。
有益效果
本发明建立了无血清无动物源性物质的光感受器神经元的诱导培养体系。因此，使用多种来源的成体细胞(例如人源)经过重编程及定向分化的方法得到了光感受器神经元，且具有各项完备的生物学功能，多个批次之间状态稳定，纯度高，解决了细胞类药物生产过程中纯度低，周期长的问题；而且本发明同时解决了长久以来动物源性培养方法和滋养细胞污染的问题，能够用于多种视觉障碍疾病的移植治疗，因此具有巨大的经济效应和社会效应。
具体实施方式
以下通过具体实施例详细说明本发明的实施过程和产生的有益效果，旨在帮助阅读者更好地理解本发明的实质和特点，不作为对本案可实施范围的限定。
实施例1.外胚层细胞诱导培养基的制备
诱导培养基的配方，以下简称NouvNeu-basal：
杜氏改良伊格尔/F12培养基(DMEM/F12培养基，SIGMA)，0.1％-2％最低必须培养基非必需氨基酸(Minimum Essential Medium Non-Essential Amino Acids，货号：11140076，Thermo Fisher)，0.1-0.8g/L氯化钠(Sodium Chloride，0.5g/L,SIGMA)，0.1-80mg/L维生素C(L-ascorbic acid，64mg/L，SIGMA)，0.1-1.8uM维生素B12(1.2uM，SIGMA)，0.1-9.8ng/ml黄体酮(Progesterone，6.3ng/ml，SIGMA)，10-35ug/ml腐胺(Putrescine，23ug/ml，SIGMA)， 1ug/L-25ug/L亚硒酸钠(最佳浓度为13.6ug/L，SIGMA)，0.1-44ug/ml胰岛素(22ug/ml,Solarbio)，0.5-20umol/L Optiferrin(10μmol/L，SIGMA)，2.5-100ng/ml植物源重组人碱性生长因子(OsrbFGF，最佳浓度为50ng/mL，禾元生物)。
本发明外胚层细胞诱导培养基的制备为在NouvNeu-basal基础培养基中加入55nM-25uM LY2157299(Selleck)(最佳浓度为7.5uM)(优选为100nM、300nM、500nM、700nM、900nM、1uM、2.5uM、5uM、7.5uM、10uM、12.5uM、15uM、17.5uM、20uM或22uM)。以下简称LY诱导方法。
对照实验为，NouvNeu basal基础培养基中添加100nM LDN-193189(Selleck)和10uM SB431542(Selleck)，以下简称LSB诱导方法。
实施例2.外胚层的诱导与鉴定
2.1外胚层的化学诱导
外胚层细胞来源于人多能干细胞，包括胚性多能干细胞，如H9细胞系和人诱导多能干细胞。其中人诱导多能干细胞按照“重编程培养基及重编程诱导多能干细胞的培养方法”(ZL201910050800.7)进行从CD34+细胞重编程获得。在本发明中，“诱导多能干细胞”和“多能干细胞”可以互换使用。
人多能干细胞使用Matrigel(STEMCELL Technologies)包被T25细胞培养瓶，铺板后置于37℃恒温箱中孵育一个小时以上。按照1x10 6细胞接种于T25培养瓶中进行扩增和传代。
进行外胚层细胞诱导时，使用50ug/ml多聚赖氨酸(SIGMA，P6407)包被6孔培养板，铺板后置于37℃恒温箱中孵育3小时以上；然后使用5ug/ml层粘连蛋白(Laminin，I2020,SIGMA ALDRICH)进一步包被，铺板后置于37℃恒温箱中孵育3小时以上。当多能干细胞达到70％的覆盖率时，用EDTA在37℃消化5分钟，使用DMEM终止细胞消化。细胞洗涤离心后按照2×10 5每瓶的比例重新接种于T25培养板中。使用本发明神经干细胞诱导培养基进行诱导，每天更换培养基直至形成片层状外胚层细胞。
外胚层用EDTA在37℃消化5分钟，使用DMEM终止细胞消化。细胞洗涤离心后按照2×10 5每瓶的比例重新接种于50ug/ml多聚赖氨酸及5ug/ml层粘连蛋白包被的T25培养板中，进行扩增培养。培养条件为37℃，5％的CO 2(Panasonic，MCO-18AC)。
2.2外胚层细胞的鉴定
外胚层细胞可以进一步形成神经干细胞，为了鉴定外胚层细胞的这一能力，在体外使用NouvNeu-basal基础培养基，添加7.5uM LY2157299和0.5-20uM JW55(最佳浓度为5uM)，进一步培养实施例2.1中得到的外胚层细胞，直至形成神经花环状结构。使用LSB诱导方法作为对照。
分别取LY诱导方法和LSB诱导方法得到的外胚层分化得到的神经干细胞进行免疫荧光染色鉴定：采用4％多聚甲醛室温固定细胞40分钟，用DPBS缓冲液清洗两遍；然后用0.1％Triton X-100透化处理5分钟，用DPBS缓冲液清洗两遍；然后用含10％马血清和0.1％Triton X-100的DPBS缓冲液将细胞4℃孵育过夜；然后加入用DPBS缓冲液稀释的抗体，37℃孵育2小时，用DPBS缓冲液清洗三遍后拍照，拍照使用Leica DMi8进行。抗体使用细节如表1所示。结果如图1a-1d所示，显示LY诱导方法和对照LSB诱导方法均能诱导具有神经花环结构的神经干细胞产生，且得到的神经干细胞表达Pax6和Nestin。
表1.细胞免疫荧光染色所用抗体
Figure PCTCN2020139521-appb-000001
使用Q-PCR检测从外胚层细胞向神经干细胞诱导过程中的不同标志物基因的转录变化。实施例2中使用的外胚层细胞，使用LY诱导方法和LSB诱导对照方法得到的神经干细胞，分别使用RNeasy Mini or Micro Kit(QIAGEN)进行总RNA抽提，1mg RNA用SuperScript III First-Strand Synthesis System(Invitrogen)合成cDNA。用SYBR Premix Ex Taq(TaKaRa)和Thermal Cycler Dice Real Time System(TaKaRa)来进行Quantitative PCR的标记和反应，beta-Actin用来作为内参。所有数据用delta-Ct method进行分析。每组试验使用三组重复进行试验，并进行方差统计。用于鉴定不同细胞标志物的编码基因的引物序列如表2所示。结果如图1e-1g所示。结果显示LY诱导方法和对照LSB诱导方法诱导得到的神经干细胞在转录水平表达多种神经干细胞标志物SOX2、PAX6和Nestin。因此，本发明获得的外胚层具有进一步分化为神经干细胞的能力。
2.3不同浓度LY2157299对外胚层细胞的转录水平的影响
使用Q-PCR检测不同浓度LY2157299对外胚层细胞形成过程中的影响。实施例2中使用的诱导多能干细胞，使用LY诱导方法中不同浓度LY2157299和LSB诱导方法得到的外胚层细胞，分别使用RNeasy Mini or Micro Kit(QIAGEN)进行总RNA抽提，1mg RNA用SuperScript III First-Strand Synthesis System(Invitrogen)合成cDNA。用SYBR Premix Ex Taq(TaKaRa)和Thermal Cycler Dice Real Time System(TaKaRa)来进行Quantitative PCR的标记和反应，beta-Actin用来作为内参。所有数据用delta-Ct method进行分析。每组试验使用三组重复进行试验，并进行方差统计。用于鉴定不同细胞标志物的编码基因的引物序列如表2所示。如图2所示，结果显示无LY2157299或者低浓度情况下，外胚层不能充分形成，高浓度则引发细胞凋亡，因此本发明使用的浓度为LY2157299最佳浓度范围。
表2.外胚层及神经干细胞检测引物
SOX2-F CATGCAGGTTGACACCGTTGG(SEQ ID NO:1)
SOX2-R ATGGATTCTCGGCAGACTGATTCA(SEQ ID NO:2)
PAX6-F TCTTTGCTTGGGAAATCCG(SEQ ID NO:3)
PAX6-R CTGCCCGTTCAACATCCTTAG(SEQ ID NO:4)
Nestin-F TCAAGATGTCCCTCAGCCTGGA(SEQ ID NO:9)
Nestin-R TGGCACAGGTGTCTCAAGGGTAG(SEQ ID NO:10)
CASP3-F GGAAGCGAATCAATGGACTCTGG(SEQ ID NO:5)
CASP3-R GCATCGACATCTGTACCAGACC(SEQ ID NO:6)
ACTIN-F TCCCTGGAGAAGAGCTACGA(SEQ ID NO:7)
ACTIN-R AGCACTGTGTTGGCGTACAG(SEQ ID NO:8)
实施例3.外胚层细胞分化为光感受器神经元细胞
将实施例2中NouvNeu系统获得的外胚层细胞在NouvNeu培养基中进行定向分化。进行分化时，使用50ug/ml多聚赖氨酸(SIGMA，P6407)包被6孔培养板，铺板后置于37℃恒温箱中孵育3小时以上，直至细胞接种。
本发明光感受器神经元细胞诱导培养基的制备为在NouvNeu-basal基础培养基中加入55nM-25uM LY2157299(最佳浓度为7.5uM)(优选为100nM、300nM、500nM、700nM、900nM、1uM、2.5uM、5uM、7.5uM、10uM、12.5uM、15uM、17.5uM、20uM或22uM)以及0.05-0.95uM AM580(Selleck)(最佳浓度为0.5uM)(优选为0.08uM，0.09uM，0.1uM，0.2uM，0.3uM，0.5uM，0.6uM，0.7uM，0.8uM，0.9uM)。直至细胞形成光感受器神经元细胞。在NouvNeu培养基中添加0.5uM视黄酸(Retinoic acid)形成对照实验，以下简称RA。
AM580浓度筛选试验参照实施例2.3进行，比较使用AM580诱导方法中不同浓度AM580和LSB诱导方法得到的光感受器神经元细胞的表达差异。引物使用列表见表2及表4。如图3所示，结果表明AM580浓度与光感受器非特异性标志物PAX6呈正相关，但当AM580浓度过高时，会引起细胞凋亡。结合图4中AM580对光感受器神经元特异性基因的表达，因此，本发明使用的AM580浓度为最佳使用浓度。
每隔3天更换新鲜培养基，直至第21天。培养条件为37℃，5％的CO 2(Panasonic，MCO-18AC)。诱导完成后，分别取每组对照两种材料进行免疫荧光染色鉴定：采用4％多聚甲醛室温固定细胞40分钟，用DPBS缓冲液清洗两遍；然后用0.1％Triton X-100透化处理5分钟，用DPBS缓冲液清洗两遍；然后用含10％马血清和0.1％Triton X-100的DPBS缓冲液将细胞4℃孵育过夜；然后加入2％马血清和0.1％Triton X-100的DPBS缓冲液稀释的一抗，37℃孵育2小时，用DPBS缓冲液清洗三遍后，加入2％马血清和0.1％Triton X-100的DPBS缓冲液稀释的二抗拍照。抗体使用细节如表3所示。实验结果表明实施例3中得到的光感受器神经元和RA对照一样，具有轴突结构且表达光感受器神经元标志物OPSIN、CRX、RECOVERIN、RHODOPSIN和PAX6，以及神经谱系标志物Nestin(图5)。
表3.定向分化免疫荧光染色所用抗体
Figure PCTCN2020139521-appb-000002
使用Q-PCR检测从外胚层细胞向光感受器神经细胞诱导过程中的不同标志物基因的转录变化。使用本发明及不同浓度AM580获得的光感受器细胞，以及RA对照诱导方法得到的光感受器细胞，分别使用RNeasy Mini or Micro Kit(QIAGEN)进行总RNA抽提，1mg RNA用SuperScript III First-Strand Synthesis System(Invitrogen)合成cDNA。用SYBR Premix Ex Taq(TaKaRa)和Thermal Cycler Dice Real Time System(TaKaRa)来进行Quantitative PCR的标记和反应，beta-Actin用来作为内参。所有数据用delta-Ct method进行分析。每组试验使用三组重复进行试验，并进行方差统计。用于鉴定不同细胞标志物的编码基因的引物序列如表4所示。结果显示，使用AM580可以诱导细胞表达多种光感受器细胞特异标志基因，且不显著低于RA对照(图4)。
表4.光感受器细胞诱导过程中的不同标志物基因引物
CRX-F CTTGTAGAGGACGCAGTCTCCATCT(SEQ ID NO:11)
CRX-R CAATCGTGCCAGACGGTGTTAGG(SEQ ID NO:12)
OPN1LW-F TCATCGCCAGCACTATCAGCATTG(SEQ ID NO:13)
OPN1LW-R ACAGCAGACCAGATCCAGGAGAAG(SEQ ID NO:14)
Recoverin-F CAGTTCCAGAGCATCTACGCCAAG(SEQ ID NO:15)
Recoverin-R GCCATGACGATCTCCAGCACTTC(SEQ ID NO:16)
Rhodopsin-F CAGCGTGGCATTCTACATCTTCAC(SEQ ID NO:17)
Rhodopsin-R ACCTGGCTCGTCTCCGTCTT(SEQ ID NO:18)
PAX6-F TCTTTGCTTGGGAAATCCG(SEQ ID NO:19)
PAX6-R CTGCCCGTTCAACATCCTTAG(SEQ ID NO:20)
βActin-F GGCCGAGGACTTTGATTGCACA(SEQ ID NO:21)
βActin-R GGGCACGAAGGCTCATCATTCAA(SEQ ID NO:22)
实施例4.基础培养体系中关键成分的浓度鉴定：
本发明实施例中示出的NouvNeu-basal基础培养基的配方为杜氏改良伊格尔/F12培养基(DMEM/F12培养基)、0.1％-2％最低必须培养基非必需氨基酸(Minimum Essential Medium Non-Essential Amino Acids，最佳浓度为1％)、0.1-0.8g/L氯化钠(Sodium Chloride，最佳浓度为0.5g/L)、0.1-80mg/L维生素C(L-ascorbic acid，最佳浓度为64mg/L)、0.1-1.8uM维生素B12(最佳浓度为1.2uM)、0.1-9.8ng/ml黄体酮(Progesterone，最佳浓度6.3ng/ml)、10-35ug/ml腐胺(Putrescine，最佳浓度为23ug/ml)、1ug/L-25ug/L亚硒酸钠(最佳浓度为13.6ug/L)、0.1-44ug/ml胰岛素(最佳浓度为22ug/ml)、0.5-20umol/L Optiferrin(最佳浓度为10μmol/L)、2.5-100ng/ml植物源重组人碱性生长因子(OsrbFGF，最佳浓度为50ng/mL)。
在保持其他成分最佳浓度的情况话，取氯化钠的不同浓度梯度配制筛选培养体系。使用全自动冰点渗透压计(FM-8P，上海医大仪器有限公司)来检测不同浓度氯化钠对培养体系渗透压的影响，具体操作参加产品说明书(FM-8P，上海医大仪器有限公司)。试验结果详见图6a，结果说明氯化钠浓度与培养体系渗透压呈线性增加。但当浓度达到1g/ml时，培养体系的渗透压已经超过细胞培养极限渗透压320mOsm/kg。说明本发明使用的氯化钠浓度为最适于细胞培养的浓度。
维生素B12浓度筛选试验参照实施例2.3进行，比较使用不同浓度维生素B12得到的光感受器神经元细胞的表达差异。引物使用列表见表2及表4。如图6b所示，结果表明维生素B12浓度与光感受器非特异性标志物PAX6呈正相关，但当维生素B12浓度过高时，引起细胞非特异性分化，从而导致PAX6相对表达量降低。因此，本发明使用的维生素B12浓度为最佳使用浓度。
在保持其他成分最佳浓度的情况话，分别取胰岛素或者维生素C的不同浓度梯度配制筛选培养体系。按照实施例2.1的方法进行外胚层诱导。使用Cyquant试验来进行细胞活力定量检测，从而研究胰岛素或者维生素C在细胞长期培养过程中对细胞生理状态的影响。包被96孔不透光细胞培养板，包被完成后将细胞按照5x10 4每孔的细胞数量分别接种，并设置三组平行重复(三组的平均值作为数据进行计算)，重复实施例2.1的过程，培养条件为37℃，5％二氧化碳。分别在1天、5天、10天以及20天取样，细胞活力检测使用CyQuant Kit(Invitrogen,货号:X12223)进行，按照使用说明书操作，使用SpectraMax i3 Multi-Mode Microplate Reader(VWR，型号ID3-STD)进行数据读取。结果如图6c-6d所示，Cyquant实验显示，诱导得到的细胞数量与胰岛素或者维生素C均称正相关，但是在高浓度下，细胞数量与本发明中的最佳浓度相比，差异不显著；此外，高浓度维生素C在诱导后期导致了细胞成片脱落，不能继续进行诱导。因此，结合成本因素，本发明基础培养基中的胰岛素或者维生素C为最佳浓度。
同样使用Cyquant试验来进行黄体酮，腐胺和Optiferrin对细胞活力的检测，从而研究不 同浓度黄体酮，腐胺和Optiferrin在光感受器细胞诱导过程中对细胞生理状态的影响，结果如图6e，6f，和图6g所示，黄体酮，腐胺和Optiferrin浓度均与细胞增殖速度呈正相关，结合成本因素，本发明使用的黄体酮，腐胺和Optiferrin范围属于最佳使用范围。
需要说明的是，上述实施例仅为本发明较佳的具体实施方式，并不以任何形式限制本发明，凡根据本发明的技术方案等同替换或改变获得的技术方案，均涵盖在本发明的保护范围内。

Claims (20)

  1. LY2157299在多能干细胞分化为外胚层中的应用，优选地，所述LY2157299的用量为55nM-25uM，更优选为100nM、300nM、500nM、700nM、900nM、1uM、2.5uM、5uM、7.5uM、10uM、12.5uM、15uM、17.5uM、20uM或22uM。
  2. 培养基，其特征在于，在NouvNeu-basal基础培养基中加入LY2157299，所述NouvNeu-basal基础培养基的配方为杜氏改良伊格尔/F12培养基(DMEM/F12培养基)、0.1％-2％最低必须培养基非必需氨基酸(Minimum Essential Medium Non-Essential Amino Acids，最佳浓度为1％)、0.1-0.8g/L氯化钠(Sodium Chloride，最佳浓度为0.5g/L)、0.1-80mg/L维生素C(L-ascorbic acid，最佳浓度为64mg/L)、0.1-1.8uM维生素B12(最佳浓度为1.2uM)、0.1-9.8ng/ml黄体酮(Progesterone，最佳浓度6.3ng/ml)、10-35ug/ml腐胺(Putrescine，最佳浓度为23ug/ml)、1ug/L-25ug/L亚硒酸钠(最佳浓度为13.6ug/L)、0.1-44ug/ml胰岛素(最佳浓度为22ug/ml)、0.5-20umol/L Optiferrin(最佳浓度为10μmol/L)、2.5-100ng/ml植物源重组人碱性生长因子(OsrbFGF，最佳浓度为50ng/mL)。
  3. 根据权利要求2所述的培养基，其特征在于，所述LY2157299的用量为55nM-25uM，优选为100nM、300nM、500nM、700nM、900nM、1uM、2.5uM、5uM、7.5uM、10uM、12.5uM、15uM、17.5uM、20uM或22uM。
  4. 根据权利要求3所述的培养基，其特征在于，所述LY2157299的用量为7.5uM。
  5. 将多能干细胞(优选人源多能干细胞)分化为外胚层的方法，其特征在于，将多能干细胞在根据权利要求2-4任一项所述的培养基中培养，获得外胚层细胞。
  6. 培养基，其特征在于，在NouvNeu-basal基础培养基中加入LY2157299和JW55，所述NouvNeu-basal基础培养基的配方为杜氏改良伊格尔/F12培养基(DMEM/F12培养基)、0.1％-2％最低必须培养基非必需氨基酸(Minimum Essential Medium Non-Essential Amino Acids，最佳浓度为1％)、0.1-0.8g/L氯化钠(Sodium Chloride，最佳浓度为0.5g/L)、0.1-80mg/L维生素C(L-ascorbic acid，最佳浓度为64mg/L)、0.1-1.8uM维生素B12(最佳浓度为1.2uM)、0.1-9.8ng/ml黄体酮(Progesterone，最佳浓度6.3ng/ml)、10-35ug/ml腐胺(Putrescine，最佳浓度为23ug/ml)、1ug/L-25ug/L亚硒酸钠(最佳浓度为13.6ug/L)、0.1-44ug/ml胰岛素(最佳浓度为22ug/ml)、0.5-20umol/L Optiferrin(最佳浓度为10μmol/L)、2.5-100ng/ml植物源重组人碱性生长因子(OsrbFGF，最佳浓度为50ng/mL)；任选地，所述JW55的浓度为0.5-20uM，最佳浓度为5uM；任选地，所述LY2157299的用量为55nM-25uM，优选为100nM、300nM、500nM、700nM、900nM、1uM、2.5uM、5uM、7.5uM、10uM、12.5uM、15uM、17.5uM、20uM或22uM。
  7. 将多能干细胞(优选人源多能干细胞)分化为神经干细胞的方法，其特征在于，将多能干细胞在根据权利要求6所述的培养基中培养。
  8. AM580在多能干细胞(优选人源多能干细胞)分化为光感受器神经元细胞中的应用，优选地，所述AM580的用量为0.05-0.95μM，更优选为0.08uM，0.09uM，0.1uM，0.2uM，0.3uM，0.5uM，0.6uM，0.7uM，0.8uM，0.9uM。
  9. AM580在外胚层细胞分化为光感受器神经元细胞中的应用，优选地，所述AM580的用量为0.05-0.95μM，更优选为0.08uM，0.09uM，0.1uM，0.2uM，0.3uM，0.5uM，0.6uM，0.7uM，0.8uM，0.9uM。
  10. LY2157299和AM580在多能干细胞(优选人源多能干细胞)分化为光感受器神经元细胞中的应用，优选地，所述AM580的用量为0.05-0.95μM，优选为0.08uM，0.09uM，0.1uM，0.2uM，0.3uM，0.5uM，0.6uM，0.7uM，0.8uM，0.9uM。
  11. 根据权利要求10所述的应用，其中所述LY2157299的用量为55nM-25uM，优选为100nM、300nM、500nM、700nM、900nM、1uM、2.5uM、5uM、7.5uM、10uM、12.5uM、15uM、17.5uM、20uM或22uM。
  12. 光感受器神经元细胞的诱导培养基，其特征在于：在NouvNeu-basal基础培养基中加入55nM-25uM LY2157299(优选为100nM、300nM、500nM、700nM、900nM、1uM、2.5uM、5uM、7.5uM、10uM、12.5uM、15uM、17.5uM、20uM或22uM)以及0.05-0.95uM AM580(优选为0.08uM，0.09uM，0.1uM，0.2uM，0.3uM，0.5uM，0.6uM，0.7uM，0.8uM，0.9uM)，所述NouvNeu-basal基础培养基的配方为杜氏改良伊格尔/F12培养基(DMEM/F12培养基)、0.1％-2％最低必须培养基非必需氨基酸(Minimum Essential Medium Non-Essential Amino Acids，最佳浓度为1％)、0.1-0.8g/L氯化钠(Sodium Chloride，最佳浓度为0.5g/L)、0.1-80mg/L维生素C(L-ascorbic acid，最佳浓度为64mg/L)、0.1-1.8uM维生素B12(最佳浓度为1.2uM)、0.1-9.8ng/ml黄体酮(Progesterone，最佳浓度6.3ng/ml)、10-35ug/ml腐胺(Putrescine，最佳浓度为23ug/ml)、1ug/L-25ug/L亚硒酸钠(最佳浓度为13.6ug/L)、0.1-44ug/ml胰岛素(最佳浓度为22ug/ml)、0.5-20umol/L Optiferrin(最佳浓度为10μmol/L)、2.5-100ng/ml植物源重组人碱性生长因子(OsrbFGF，最佳浓度为50ng/mL)。。
  13. 根据权利要求12所述的光感受器神经元细胞诱导培养基，其特征在于：所述LY2157299的用量为7.5uM，所述AM580的用量为0.5uM。
  14. 多能干细胞(优选人源多能干细胞)分化为光感受器神经元细胞的方法，其特征在于，将多能干细胞在根据权利要求2-4任一项所述的培养基中培养，获得外胚层细胞，然后将外胚层细胞在根据权利要求10-11任一项所述的光感受器神经元细胞诱导培养基中培养，获得光感受器神经元细胞，优选地，所述培养是在有基底膜的情况下进行的，更优选地，所述基底膜为基底胶(Matrigel)、层粘蛋白(Laminin)和玻连蛋白的一种或多种的组合。
  15. 根据权利要求14所述的多能干细胞分化为光感受器神经元细胞的方法，其特征在于，所述LY2157299的用量为7.5uM。
  16. 外胚层细胞，其特征在于利用权利要求2-4任一项所述的培养基培养或利用权利要求5所述的方法制备。
  17. 光感受器神经元细胞，其特征在于利用权利要求12-13任一项所述的诱导培养基培养或利用权利要求14所述的方法制备或利用权利要求14所述的外胚层细胞制备。
  18. 权利要求17所述的光感受器神经元细胞在视觉神经的退行性疾病的治疗中的用途，所述视觉神经的退行性疾病例如是与年龄相关的黄斑变性、色素变性。
  19. 权利要求17所述的光感受器神经元细胞在光感受器神经细胞移植中的用途。
  20. 一种试剂盒，其包含NouvNeu-basal基础培养基，LY2157299，和/或AM580，优选地，所述LY2157299的用量为55nM-25uM优选为100nM、300nM、500nM、700nM、900nM、1uM、2.5uM、5uM、7.5uM、10uM、12.5uM、15uM、17.5uM、20uM或22uM，所述AM580的用量为0.05-0.95uM，优选为0.08uM，0.09uM，0.1uM，0.2uM，0.3uM，0.5uM，0.6uM，0.7uM，0.8uM，0.9uM。
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