WO2015143622A1 - 一种建立神经上皮干细胞的培养基、方法及其应用 - Google Patents

一种建立神经上皮干细胞的培养基、方法及其应用 Download PDF

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WO2015143622A1
WO2015143622A1 PCT/CN2014/074029 CN2014074029W WO2015143622A1 WO 2015143622 A1 WO2015143622 A1 WO 2015143622A1 CN 2014074029 W CN2014074029 W CN 2014074029W WO 2015143622 A1 WO2015143622 A1 WO 2015143622A1
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stem cells
medium
neuroepithelial
neuroepithelial stem
cell
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French (fr)
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李天晴
季维智
李博
朱小庆
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云南中科灵长类生物医学重点实验室
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Priority to PCT/CN2014/074029 priority Critical patent/WO2015143622A1/zh
Priority to US14/770,283 priority patent/US20160251612A1/en
Priority to CN201480002707.4A priority patent/CN105121633B/zh
Publication of WO2015143622A1 publication Critical patent/WO2015143622A1/zh

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Definitions

  • the present invention relates to the field of cell biology, and in particular to an established neuroepithelial Stem cell culture medium, method and application thereof.
  • Neurological diseases are major diseases that threaten human health. Most of these diseases are caused by Neuronal cells are caused by irreplaceable loss, and traditional treatment methods are difficult to treat. Treatment, and the repair and regeneration of neural stem cells provide the possibility to cure these diseases.
  • Neuroepithelial stem cells undergo two distinct developmental stages during brain development. Neuroepithelial stem cells, radioactive glial precursor cells (radial glial progenitor cells). Neuroepithelial stem cells are the earliest neural stem cells. It has stronger differentiation pluripotency and can differentiate into whole brain cells. Neuroepithelial Stem cells have a strong value-added ability to produce high-purity neurons with important science And clinical application value.
  • the conventional neural stem cell culture method mainly adopts growth.
  • the combination of factor bFGF and EGF, this culture system can only cultivate the gods in the late stage of development.
  • the precursor cells radio-glial precursor cells
  • the nerve epithelium for a long time
  • cell and culture of radioactive glial precursor cells with prolonged culture time, cell characteristics And the pluripotency of differentiation produces significant changes, losing the ability of neural cells to differentiate, so Can not carry out large-scale production of stem cells, limiting its clinical stem cell replacement therapy, disease Mechanisms and applications in drug screening.
  • the object of the present invention is to provide a culture medium and a method for establishing neural epithelial stem cells. And its application to solve the above problems.
  • a medium for establishing a neural epithelial stem cell include;
  • the differentiation The medium includes: Neurobasal medium, B-27 additive, N-2 additive, bFGF, Agonist of Wnt signaling pathway (inhibitor of GSK3), inhibition of TGF- ⁇ signaling pathway Agents, Notch signaling pathway inhibitors, inhibitors of ALK2 and ALK3 signaling pathways;
  • the expansion medium includes: Neurobasal medium, B-27 additive, N-2 additive, bFGF, agonist of Wnt signaling pathway (GSK3 inhibitor), TGF- ⁇ signaling pathway Inhibitors and leukemia inhibitors.
  • the medium for establishing neuroepithelial stem cells includes differentiation culture Base and expansion medium
  • differentiation medium contains a variety of pluripotent stem cells for differentiation into primary Nutrients required for neuroepithelial stem cells; among them, bFGF can promote neural stem cells Early differentiation and proliferation, activation of the Wnt signaling pathway (through the activation of the Wnt signaling pathway) Mobilizer) is beneficial to the proliferation of neural stem cells, inhibits TGF- ⁇ signaling pathway and promotes pluripotent Directional differentiation of cells into nerve cells, inhibition of Notch signaling pathway can be rapidly promoted Directional differentiation of pluripotent stem cells into neurons and neural stem cells to neurons, ALK2 And inhibition of the ALK3 signaling pathway will inhibit BMP4 signaling pathway and promote pluripotent stem cells Directed differentiation of neurons and neural stem cells into neurons.
  • pluripotent stem cell differentiation can be achieved by a differentiation medium containing the above composition.
  • a differentiation medium containing the above composition.
  • amplification culture bFGF an agonist of the Wnt signaling pathway (GSK3 inhibitor)
  • TGF- ⁇ Inhibitors of signaling pathways have the primary neuroepithelial stem cells retain their properties and are stable The function of amplification; in addition, leukemia inhibitory factor has a function of regulating cell proliferation and differentiation.
  • Primary neuroepithelial stem cells can be cultured to achieve stable expansion and survival by amplifying the medium. And neuroepithelial stem cells capable of large-scale production.
  • the agonist of the Wnt signaling pathway is CHIR99021; the inhibitor of the TGF- ⁇ signaling pathway is SB431542; the Notch signaling pathway inhibitor is Compound E; the ALK2 And the inhibitor of the ALK3 signaling pathway is LDN193189;
  • the agonist of the Wnt signaling pathway and the The GSK3 inhibitors are both CHIR99021; the inhibitor of the TGF- ⁇ signaling pathway is SB431542.
  • the concentration of the bFGF is 3-100 ng/ml;
  • the concentration of the CHIR99021 is 0.3-30 ⁇ M/l, and the concentration of the SB431542 is 2-50 ⁇ M / l;
  • the concentration of the Compound E is 0.05-10 ⁇ M / l;
  • the LDN193189 The concentration is 0.1-10 ⁇ M / l;
  • the concentration of the bFGF is 3-100 ng/ml
  • the concentration of the CHIR99021 is 0.3-30 ⁇ M/l
  • the concentration of the SB431542 is 5-50 ⁇ M / l
  • the concentration of the leukemia inhibitory factor is 50-5000 U / L.
  • the invention also provides a method for obtaining neuroepithelial stem cells by using the above medium.
  • the law includes the following steps:
  • the pluripotent stem cells are digested by collagenase into cell clumps;
  • the primary neuroepithelial stem cells are cultured using the amplification medium to obtain Neuroepithelial stem cells that can be stably passaged.
  • the primary neural epithelial stem cells are used in the step of using the amplification culture After the culture is cultured to obtain neuroepithelial stem cells that can be stably passaged, it also includes:
  • the single neuroepithelial stem cells are cultured for 14-15 days using the expansion medium.
  • a neural tube in which the single neuroepithelial stem cells self-assemble is obtained.
  • the primary neural epithelial stem cells are used in the step of using the amplification culture After the culture is cultured to obtain neuroepithelial stem cells that can be stably passaged, it also includes:
  • the neuroepithelial stem cells contain Neurobasal medium, B27, non-essential amino groups Cultured and differentiated on a neuronal differentiation medium of acid and glutamine to obtain a purity of 40%-100% of neurons.
  • the step of digesting the pluripotent stem cells into a cell mass by collagenase Medium is 5-40 minutes.
  • the primary neural epithelial stem cells are used in the expansion medium After the step of culturing to obtain neuroepitary stem cells capable of stable passage, the method further comprises: The neuroepithelial stem cells were digested and passaged with 0.05% trypsin to obtain stable Neuroepithelial stem cell line.
  • a neuroepithelial stem cell according to the above-mentioned right and using the epithelial cell culture The resulting cell lines are used in cell replacement therapy, disease mechanisms, and drug screening.
  • Example 1 is a directional representation of pluripotent stem cells differentiated into a neuroepithelial stem according to Example 2 of the present invention; Schematic diagram of cells;
  • Example 2 is a long-term scale culture of neuroepithelial stem cell culture according to Example 2 of the present invention. Raise and maintain the structure of stem cells self-assembling into neural tubes and produce high-purity neurons
  • Figure 3 is a diagram showing the self-assembly of a single neuroepithelial stem cell into a nerve according to Example 2 of the present invention. Schematic diagram of tube structure;
  • Example 4 is a self-assembly of neural epithelial stem cells into a neural tube structure according to Example 2 of the present invention; And a schematic diagram of the mechanism of transformation into a radioactive glial precursor cell;
  • Figure 5 is a simulation of a human god using a single neuroepithelial stem cell in Example 2 of the present invention. Schematic diagram of the disease, differentiation into cerebral cortical nerve cells, and neurological replacement therapy.
  • This embodiment provides a medium for establishing neural epithelial stem cells, comprising: Pluripotent stem cells induce differentiation into primary neural epithelial stem cell differentiation medium and An expansion medium for amplifying the primary neuroepithelial stem cells;
  • the differentiation medium includes: Neurobasal medium, B-27 additive, N-2 Tim Additive, bFGF, agonist of Wnt signaling pathway, inhibitor of GSK3, TGF- ⁇ signal Pathway inhibitors, Notch signaling pathway inhibitors, ALK2, and ALK3 signaling pathways Inhibitor;
  • the expansion medium comprises: Neurobasal medium, B-27 additive, N-2 additive, bFGF, agonist of Wnt signaling pathway, GSK3 inhibitor, TGF- ⁇ Inhibitors of signaling pathways and leukemia inhibitors.
  • the medium for establishing neuroepithelial stem cells includes differentiation culture Base and expansion medium
  • differentiation medium contains a variety of pluripotent stem cells for differentiation into primary Nutrients required for neuroepithelial stem cells; among them, bFGF can promote neural stem cells Early differentiation and proliferation, activation of the Wnt signaling pathway (through the activation of the Wnt signaling pathway) Mobilizer) is beneficial to the proliferation of neural stem cells, inhibits TGF- ⁇ signaling pathway and promotes pluripotent Directional differentiation of cells into nerve cells, inhibition of Notch signaling pathway can be rapidly promoted Directional differentiation of pluripotent stem cells into neurons and neural stem cells to neurons, ALK2 And inhibition of the ALK3 signaling pathway will inhibit BMP4 signaling pathway and promote pluripotent stem cells Directed differentiation of neurons and neural stem cells into neurons.
  • pluripotent stem cell differentiation can be achieved by a differentiation medium containing the above composition.
  • a differentiation medium containing the above composition.
  • amplification culture bFGF an agonist of the Wnt signaling pathway (GSK3 inhibitor)
  • TGF- Inhibitors of the beta signaling pathway have the primary neuroepithelial stem cells to maintain their properties and stabilize
  • the function of amplification in addition, leukemia inhibitory factor has the function of regulating cell proliferation and differentiation can.
  • Primary neuroepithelial stem cells can be cultured into stable amplification and storage by amplifying the culture medium. Live, as well as neuroepithelial stem cells that can be produced on a large scale.
  • the Wnt signaling pathway Both the agonist and the inhibitor of GSK3 are CHIR99021; the TGF- ⁇ signaling pathway The inhibitor is SB431542; the Notch signaling pathway inhibitor is Compound E; The inhibitor of ALK2 and ALK3 signaling pathway is LDN193189;
  • the agonist of the Wnt signaling pathway and the GSK3 inhibition The preparation is CHIR99021; the inhibitor of the TGF- ⁇ signaling pathway is SB431542.
  • the concentration of the bFGF is 3-100 ng / ml;
  • the concentration of the CHIR99021 is 0.3-30 ⁇ M/l, and the concentration of the SB431542 is 2-50 ⁇ M / l;
  • the concentration of the Compound E is 0.55-10 ⁇ M / l;
  • the LDN193189 The concentration is 0.1-10 ⁇ M/l;
  • the concentration of the bFGF is 3-100 ng/ml, the concentration of the CHIR99021 is 0.3-30 ⁇ M/l; the SB431542
  • the concentration is 5-50 ⁇ M/l;
  • the concentration of the leukemia inhibitory factor is 50-5000 U/l.
  • Example 1 Neurobasal medium, B-27 additive, N-2 additive, 3 ng/ml bFGF, 0.3 ⁇ M/l CHIR99021, 2 ⁇ M/l SB431542, 0.05 ⁇ M/l Compound E And 0.1 ⁇ M/l LDN193189;
  • Example 2 Neurobasal medium, B-27 additive, N-2 additive, 100 ng/ml bFGF, 30 ⁇ M/l CHIR99021, 50 ⁇ M/l SB431542, 10 ⁇ M/l Compound E And 10 ⁇ M/l LDN193189;
  • Example 3 Neurobasal medium, B-27 additive, N-2 additive, 10 ng/ml bFGF, 3 ⁇ M/l CHIR99021, 5 ⁇ M/l SB431542, 0.2 ⁇ M/l Compound E And 0.1 ⁇ M/l LDN193189.
  • Example 1 Neurobasal medium, B-27 additive, N-2 additive, 3 ng/ml bFGF, 0.3 ⁇ M/l CHIR99021, 5 ⁇ M/l SB431542 and 50U/L leukemia inhibition factor;
  • Example 2 Neurobasal medium, B-27 additive, N-2 additive, 100 ng/ml bFGF, 30 ⁇ M/l CHIR99021, 50 ⁇ M/l SB431542 and 5000U/L leukemia Factor
  • Example 3 Neurobasal medium, B-27 additive, N-2 additive, 10 ng/ml bFGF, 3 ⁇ M/l CHIR99021, 5 ⁇ M/l SB431542 and 1000U/l leukemia inhibitory factor child.
  • the present invention is also in the above-mentioned first embodiment Based on the second embodiment, the second embodiment establishes a nerve according to the first embodiment of the medium.
  • the method of epithelial stem cells is now elaborated and explained in detail, please refer to Figure 1 - Figure 5:
  • a method for obtaining neuroepithelial stem cells using the medium of claim 1 Including the following steps:
  • the pluripotent stem cells are digested by collagenase into cell clumps;
  • the cultured feeder layer or the feeder-free pluripotent The cells are digested with collagenase (5-40 minutes) into small pieces of stem cell mass (50-100 cells).
  • pluripotent stem cells including embryonic stem cells and induced pluripotent stems Cell.
  • the suspension cell mass is in a differentiated medium in a low-attached culture dish.
  • the suspension culture was carried out for differentiation for 6 consecutive days, and the solution was changed every 2 days. 4th in differentiation At the beginning of the day, two layers of neuroepithelial structures appear in the embryoid body, please refer to Figure 1. Will differentiate Six days of neuroepithelial structure collection (stand for 5-10 minutes, remove supernatant).
  • pluripotent stem cells are induced to differentiate into high purity neuroepithelial cells.
  • Stem cell process is the day after differentiation of pluripotent stem cells in differentiation medium Forming a typical embryoid body;
  • B-C on the 5th-6th day of differentiation, the embryoid body forms a neuroepithelial Structure, expressing Nestin.
  • D-L for embryoid bodies on days 2, 5, and 6 after differentiation, respectively Frozen sections were taken and then stained with Oct4, Sox2, Nestin and Pax6 respectively. Positive cell staining results are shown.
  • M is the Sox2, Oct4 and Quantitative changes in the number of Pax6 positive cells (**P>0.01 indicates significant changes).
  • (O-T) is a long-term cultured neuroepithelial stem cell Significant proteins that express stem cells, such as Sox2, Pax6, Nestin, and N-cadherin, but not The marker protein GFAP, neuronal protein Tuj1(S) and human expressing the pro-glial precursor cells Precursor cell marker protein Tbr2 in the lateral side of the lateral cerebral cortex of the cerebral cortex 1 is shown in T).
  • the primary neuroepithelial stem cells are cultured using the expansion medium, Obtaining neuroepithelial stem cells capable of stable passage;
  • the neuroepithelial stem cells need to be cultured continuously for 7-8 days on the culture plate. Without passing through. These passaged cells maintained exponential growth even after 50 generations. It still maintains a vigorous growth rate and its growth ability is not affected at all. Flow through Cytocytometry detects these cells and finds that they stably express stem cells during long-term culture. Marker proteins such as Nestin and Sox2. And these long-term subcultured cells remain The characteristics of differentiation into high-purity nerve cells and the characteristics of the formation of neural tube structures. Please refer to figure 2:
  • the neuroepithelial stem cell culture system can be cultured in a long-term scale.
  • Neuroepithelial stem cells and keep stem cells self-assembled into neural tube structures and high purity The production of neurons.
  • A is the embryoid body of the sixth day when cultured in laminin On the plate, a double layer of neuroepithelial structure was formed after 3 days.
  • B Differentiation for low density The obtained neuroepithelial stem cells form a structure of a rosette (neural rosettes).
  • C is In the case of high density, neuroepithelial stem cells form a neural tube structure.
  • (F-G) is a cleavage marker protein phospho-vimentin (p-vimentin) staining showed that the dividing cells were mainly located on the surface of the neural tube, while the BrdU standard The cells of the DNA synthesis phase (S phase) are located on the back of the neural tube, indicating the presence of cells Interkinetic nuclear migration. Neuroepithelial stem cells The growth curve is characterized by exponential growth as the cells pass through.
  • (I-J) is a flow cell Sorting showed that cells of the 18th and 36th generations expressed similar high ratios of Sox2 and Nestin.
  • neuroepithelial stem cells are digested and passaged with 0.05% trypsin to obtain Stable neuroepithelial stem cell line;
  • trypsin can be used for digestion and passage.
  • the pass ratio is 1:8-1:16.
  • the culture plate was previously mixed with laminin at a concentration of 5-50 ⁇ g/ml.
  • the bag is quilted for more than 2 hours, and the neuroepithelial stem can be obtained by the above-mentioned digestion and passage operation. Cell line.
  • a single neural epithelial stem cell can self-assemble into a neural tube structure, specifically , can be as follows: limited long-term culture of individual neuroepithelial stem cells Dilute and culture in a well of a 96-well plate with laminin, one cell/well, culture The nutrient base is cultured using the above expansion medium, and the solution is changed every 2-3 days until the 14th-15th Days, individual cells self-assemble into a series of neural tube structures. These single cells produce The neural tube structure expresses Sox2, Nestin, Pax6, ZO-1 and N-cadherin. BrdU Markers show that these neural tube structures have strong value-adding capabilities and possess cell division Interkinetic nuclear migration function. As shown in Figure 3.
  • FIG. 3 individual neuroepithelial stem cells are shown self-assembled into a neural tube structure.
  • A is a continuous cloning of a single neuroepithelial stem cell self-assembled into a neural tube structure.
  • schematic diagram. shows a neuroepithelial stem cell in a 96-well plate.
  • C-D is a generation A neural tube colony produced by a single neuroepithelial cell. D shows the high magnification The individual neural tube structure.
  • E-H is a single neural epithelial stem cell derived neural tube junction Expression of neuroepithelial stem cell marker proteins such as Pax6, Nestin, N-cadherin and ZO-1.
  • BrdU labeling shows that the neural tube structure of a single cell source remains strong Value-added ability.
  • J shows that live cell dynamic imaging technology indicates that neuroepithelial stem cells have a phase Like in-kind nuclear migration, and exhibits horizontal Symmetrical splitting.
  • K the percentage of clones that survived during long-term culture of neuroepithelial stem cells And the change in the proportion of polar clones that form the neural tube, the results show that the nerve epithelium is fine The increase in cell passage time, the higher the proportion of cells that produce these two types of colonies.
  • L is at Cell colony rate and surviving colony formation inoculated with cells in a continuous cloning process Comparison of neural tube polarity ratios. The results show that there is no between the first clone and the second clone. Significant difference (P>0.05).
  • the characteristics of a single cell to form a neural tube structure can be utilized.
  • a series of single cell culture experiments were performed by removing one component each time in the medium. Which components are necessary for self-renewal and neural tube formation of neuroepithelial stem cells.
  • FIG 4 it is shown that neuroepithelial stem cells self-assemble into neural tube structures and The mechanism of transformation into radioactive glial precursor cells.
  • A for individual neuroepithelial stem cells in different Comparison of cell survival and formation of polar neural tube ratios under culture conditions. *P value ⁇ 0.05 Indicates a significant difference, **P value ⁇ 0.01 indicates a very significant difference.
  • B for a single neuroepithelial Cell proliferation under different experimental conditions, with the prolongation of culture time Comparison. *P value ⁇ 0.05 indicates a significant difference.
  • C-J is the activation of the Wnt signaling pathway It is necessary for neuroepithelial stem cells to form neural tubes, and their inactivation will lose neural tube formation. The ability to convert to radioactive glial precursor cells.
  • (K-N) shows the endogenous FGFR letter The pathway is required for neural tube formation.
  • SU5402 is an inhibitor of the FGF receptor.
  • arrow Refers to the expansion of a single neuroepithelial stem cell in a neuroepithelial stem cell containing SU5402 A small neural tube structure is formed in the medium.
  • (O) self-assembly for neuroepithelial stem cells Transmembrane structure and a model transformed into a radioactive glial precursor cell.
  • leukemia inhibitory factor As can be seen from Figure 4, leukemia inhibitory factor, FGF and Wnt signaling pathways are in the gods. The role of the self-assembly process. Exogenous bFGF and leukemia inhibitors can promote God Self-renewal of epithelial stem cells and increased formation of neural tube structures, while endogenous FGFR And the Wnt signaling pathway is required for neural tube formation. In addition, the Wnt signal path is lost. In the case of living, the neuroepithelial stem cells are transformed into radioactive glial precursor cells, losing the formation of gods. The ability to manage.
  • early transformed radioactive glial precursor cells are in the Wnt signaling pathway In the case of reactivation, it can be transformed into neuroepithelial stem cells, and after passage, the radioactive gelatin The precursor cells cannot be retransformed into neuroepithelial stem cells.
  • neuroepithelial stem cells can also be utilized as well as individual Neural stem cells differentiate into high purity neurons. Long-term culture of neuroepithelial stem cells And a stable neuroepithelial stem cell line produced by a single cell, after removing bFGF, Spontaneous differentiation under conditions of CHIR99021, SB431542 and LIF, neuronal
  • the differentiation medium is Neurobasal medium, B27, non-essential amino acids (content can be 1%) And glutamine (concentration can be 1 mM).
  • Radioactive glial stem cells Day 0-3), the developmental stage of interneuronal precursor cells (days 4-6), differentiated into High-purity neurons, including excitatory glutamine-energy, inhibitory gamma-amino Butyric acid (GABA) and neurons in the II-IV layer and V-VI layer of the cerebral cortex
  • GABA inhibitory gamma-amino Butyric acid
  • the intermediate neurons of the layer are D-J in Figure 4.
  • the GFP-containing lentivirus is added to the expansion medium of neuroepithelial stem cells.
  • infected cultured neuroepithelial stem cells washed with PBS for 4 hours, replaced with Fresh medium.
  • GFP-labeled neuroepithelial stem cells as well as single GFP-tagged Stem cells by neural stem cells produced by the single cell expansion method described above The way of the instrument, in the case of anesthesia, is transplanted into the brain of the cynomolgus monkey. After 3 months, Fix the brain, perform tissue sectioning, and find that the transplanted cells can be integrated into the cynomolgus monkey.
  • the brain is divided into neurons, and these neurons grow long axons within 3 months. It is widely distributed in the cerebral cortex, and some of the axons migrate deep into the brain. therefore This cell has important clinical value and can be used for stem cell therapy of future neurological diseases. Treatment provides an important source of donor cells. As shown in Figure 5 L-O.
  • a single neural epithelial stem cell is used to simulate a human neural tube disease, And differentiated into cerebral cortical nerve cells for neuronal replacement therapy.
  • A-C shows the single The transformation of neuroepithelial stem cells into neural tubes can mimic neural tube development defects. among them, DHFR, SHMT1, MTRR, MTHFD1L, MTR and CBS are folate metabolism Key enzyme (A). These enzymes are highly expressed in neuroepithelial stem cells (B), and folic acid The concentration was positively correlated with neural tube formation and negatively correlated with colony apoptosis (C).
  • D-J A stable cell line derived from a single neuroepithelial stem cell can differentiate into a cerebral cortex Jing Yuan.
  • GABA gamma-aminobutyric acid
  • D-G excitatory glutamine and inhibitory gamma-aminobutyric acid
  • GABA inhibitory gamma-aminobutyric acid
  • K-O Single neuroepithelial cell-derived neuroepithelial stem cells were transplanted into the visual cortex of monkeys Afterwards, it can be integrated into the cerebral cortex (K); these transplanted cells are integrated into the cerebral cortex Outer layer (L-L”) and II-IV layer (M-M”), and differentiate into neurons. And these differentiated gods The axons grow very long axons, which are distributed at multiple sites in the cerebral cortex. The deep extension of the cerebral cortex is as described in O in Figure 5.
  • neuroepithelial stem cells established by the method of the present embodiment and the use of the god Cell lines produced by epithelial cell culture; and neuroepithelial stem cells and utilization of the nerve Cell lines produced by skin cell culture in cell replacement therapy, disease mechanisms, and drug screening
  • the application is also within the scope of the invention.

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Abstract

提供一种建立神经上皮干细胞的培养基、方法及其应用。所述培养基包括用于将多能干细胞诱导分化为初级神经上皮干细胞的分化培养基及用于使初级神经上皮干细胞扩增的扩增培养基。

Description

一种建立神经上皮干细胞的培养基、方法及其应用 技术领域
本发明涉及细胞生物学领域，具体而言，涉及一种建立神经上皮 干细胞的培养基、方法及其应用。
背景技术
神经性疾病是危险人类健康的重大疾病，这些疾病大多数是由于 神经细胞发生了不可替代的损失而所致，传统的治疗方法很难进行治 疗，而神经干细胞的修复再生为治愈这些疾病提供了可能。
神经干细胞在脑部的发育过程中主要经过两个不同的发育阶段， 即神经上皮干细胞（neuroepithelial stem cells）、放射胶质前体细胞 (radial glial progenitor cells)。神经上皮干细胞是最早期的神经干细胞， 具有更强的分化多能性，能分化发育为整个脑部细胞。而且神经上皮 干细胞具有很强的增值能力，产生高纯度的神经元，具有重要的科学 和临床应用价值。
但是，在相关技术中，传统的神经干细胞培养方法主要采用生长 因子bFGF和EGF组合而成，这种培养体系只能培养发育后期的神 经前体细胞（放射胶质前体细胞），而不能长期稳定培养神经上皮干 细胞，并且培养的放射胶质前体细胞随着培养时间的延长，细胞特性 以及分化的多能性产生显著的变化，失去神经细胞的分化能力，因此 不能进行干细胞的规模化生产，限制了其临床干细胞替代治疗、疾病 机制以及药物筛选中的应用。
综上，通过体外培养建立一种能够稳定传代的神经上皮干细胞是 本领域亟待解决的一个技术问题。
发明内容
本发明的目的在于提供一种建立神经上皮干细胞的培养基、方法 及其应用，以解决上述的问题。
在本发明的实施例中提供了一种建立神经上皮干细胞的培养基， 包括；
用于将多能干细胞诱导分化为初级神经上皮干细胞的分化培养 基以及用于使所述初级神经上皮干细胞扩增的扩增培养基；所述分化 培养基包括：Neurobasal培养基、B-27添加剂、N-2添加剂、bFGF、 Wnt信号通路的激动剂（GSK3的抑制剂）、TGF-β信号通路的抑制 剂、Notch信号通路抑制剂、ALK2以及ALK3信号通路的抑制剂； 所述扩增培养基包括：Neurobasal培养基、B-27添加剂、N-2添加剂、 bFGF、Wnt信号通路的激动剂（GSK3抑制剂）、TGF-β信号通路的 抑制剂和白血病抑制因子。
本发明提供的这种建立神经上皮干细胞的培养基，包括分化培养 基和扩增培养基，分化培养基中含有多种可供多能干细胞分化为初级 神经上皮干细胞所需的营养成分；其中，bFGF可以促进神经干细胞 的早期分化和增殖，Wnt信号通路的激活（通过Wnt信号通路的激 动剂）有利于神经干细胞的增殖，抑制TGF-β信号通路促进多能干 细胞往神经细胞的定向分化，Notch信号通路的抑制能够快速的促进 多能干细胞往神经细胞以及神经干细胞往神经元的定向分化，ALK2 和ALK3信号通路的抑制将抑制BMP4信号通路，促进多能干细胞 往神经细胞以及神经干细胞往神经元的定向分化。
因此，通过含有上述组成的分化培养基即可使得多能干细胞分化 成为初级神经上皮干细胞；然而初级神经上皮干细胞虽然具备神经上 皮干细胞的机能，但是其在传统的培养条件下，仍然不能长期稳定的 扩增，因此，其再通过扩增培养基进行长期稳定培养，其中，扩增培 养基中所含的bFGF、Wnt信号通路的激动剂（GSK3抑制剂）、TGF-β 信号通路的抑制剂均具备使初级神经上皮干细胞保持其特性并稳定 扩增的功能；此外，白血病抑制因子具有调节细胞增殖和分化的功能。 通过扩增培养基即可将初级神经上皮干细胞培养成稳定扩增和存活 的，以及能够规模化生产的神经上皮干细胞。
可选的，在所述分化培养基中，所述Wnt信号通路的激动剂和 GSK3的抑制剂均为CHIR99021；所述TGF-β信号通路的抑制剂为 SB431542；所述Notch信号通路抑制剂为Compound E；所述ALK2 以及ALK3信号通路的抑制剂为LDN193189；
和/或在所述扩增培养基中，所述Wnt信号通路的激动剂和所述 GSK3抑制剂均为CHIR99021；所述TGF-β信号通路的抑制剂为 SB431542。
可选的，在所述分化培养基中，所述bFGF的浓度为3-100ng/ml； 所述CHIR99021的浓度为0.3-30μM/l，所述SB431542的浓度为 2-50μM/l；所述Compound E的浓度为0.05-10μM/l；所述LDN193189 的浓度为0.1-10μM/l；
和/或在所述扩增培养基中，所述bFGF的浓度为3-100ng/ml， 所述CHIR99021的浓度为0.3-30μM/l；所述SB431542的浓度为 5-50μM/l；所述白血病抑制因子的浓度为50-5000U/L。
本发明还提供了一种利用上述培养基获得神经上皮干细胞的方 法，包括以下步骤：
将多能干细胞经过胶原酶消化成细胞团块；
将所述细胞团块悬浮在所述分化培养基中并进行悬浮培养，使其 分化为初级神经上皮干细胞；
将所述初级神经上皮干细胞使用所述扩增培养基进行培养，获得 能够稳定传代的神经上皮干细胞。
可选的，在所述步骤将所述初级神经上皮干细胞使用所述扩增培 养基进行培养，获得能够稳定传代的神经上皮干细胞之后，还包括：
将所述神经上皮干细胞进行稀释，得到单个神经上皮干细胞；
将所述单个神经上皮干细胞利用所述扩增培养基培养14-15天， 得到该单个神经上皮干细胞自我组装成的神经管。
可选的，在所述步骤将所述初级神经上皮干细胞使用所述扩增培 养基进行培养，获得能够稳定传代的神经上皮干细胞之后，还包括： 将所述神经上皮干细胞在含有Neurobasal培养基、B27、非必需氨基 酸以及谷氨酰胺的神经元分化培养基上培养并使其分化，得到纯度为 40%-100%的神经元。
可选的，在所述将多能干细胞经过胶原酶消化成细胞团块的步骤 中：所述消化时间为5-40分钟。
可选的，在所述将所述初级神经上皮干细胞使用所述扩增培养基 进行培养，获得能够稳定传代的神经上皮干细胞的步骤之后，还包括： 将所述神经上皮干细胞利用0.05%的胰酶进行消化传代，获得稳定的 神经上皮干细胞系。
一种根据上述方法建立的神经上皮干细胞以及利用该神经上皮 细胞培养产生的细胞系。
一种根据权上述神经上皮干细胞以及利用该神经上皮细胞培养 产生的细胞系在细胞替代治疗、疾病机制以及药物筛选中的应用。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方 案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简 单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式， 对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可 以根据这些附图获得其它的附图。
图1为本发明实施例2的多能干细胞定向诱导分化为神经上皮干 细胞的示意图；
图2为本发明实施例2的神经上皮干细胞培养经过长期规模化培 养，并保持干细胞自我组装为神经管的结构以及产生高纯度的神经元 的示意图；
图3为本发明实施例2的单个的神经上皮干细胞自我组装为神经 管结构示意图；
图4为本发明实施例2的神经上皮干细胞自我组装为神经管结构 以及转化为放射胶质前体细胞机制的示意图；
图5为本发明实施例2中利用单个的神经上皮干细胞模拟人的神 经管疾病，分化为大脑皮层神经细胞以及进行神经替代治疗的示意图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将对本发明 的技术方案进行清楚、完整的描述，基于本发明中的具体实施方式， 本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有 其它实施方式，都属于本发明所保护的范围。
实施例一
本实施例提供一种建立神经上皮干细胞的培养基，包括：用于将 多能干细胞诱导分化为初级神经上皮干细胞的分化培养基以及用于 使所述初级神经上皮干细胞扩增的扩增培养基；
所述分化培养基包括：Neurobasal培养基、B-27添加剂、N-2添 加剂、bFGF、Wnt信号通路的激动剂、GSK3的抑制剂、TGF-β信号 通路的抑制剂、Notch信号通路抑制剂、ALK2以及ALK3信号通路 的抑制剂；所述扩增培养基包括：Neurobasal培养基、B-27添加剂、 N-2添加剂、bFGF、Wnt信号通路的激动剂、GSK3抑制剂、TGF-β 信号通路的抑制剂和白血病抑制因子。
本发明提供的这种建立神经上皮干细胞的培养基，包括分化培养 基和扩增培养基，分化培养基中含有多种可供多能干细胞分化为初级 神经上皮干细胞所需的营养成分；其中，bFGF可以促进神经干细胞 的早期分化和增殖，Wnt信号通路的激活（通过Wnt信号通路的激 动剂）有利于神经干细胞的增殖，抑制TGF-β信号通路促进多能干 细胞往神经细胞的定向分化，Notch信号通路的抑制能够快速的促进 多能干细胞往神经细胞以及神经干细胞往神经元的定向分化，ALK2 和ALK3信号通路的抑制将抑制BMP4信号通路，促进多能干细胞 往神经细胞以及神经干细胞往神经元的定向分化。
因此，通过含有上述组成的分化培养基即可使得多能干细胞分化 成为初级神经上皮干细胞；然而初级神经上皮干细胞虽然具备神经上 皮干细胞的机能，但是其在传统的培养条件下，仍然不能长期稳定的 扩增，因此，其再通过扩增培养基进行长期稳定培养，其中，扩增培 养基中所含的bFGF、Wnt信号通路的激动剂（GSK3抑制剂）、TGF- β信号通路的抑制剂均具备使初级神经上皮干细胞保持其特性并稳 定扩增的功能；此外，白血病抑制因子具有调节细胞增殖和分化的功 能。通过扩增培养基即可将初级神经上皮干细胞培养成稳定扩增和存 活的，以及能够规模化生产的神经上皮干细胞。
更优选的，在本实施例的分化培养基中，所述Wnt信号通路的 激动剂和GSK3的抑制剂均为CHIR99021；所述TGF-β信号通路的 抑制剂为SB431542；所述Notch信号通路抑制剂为Compound E；所 述ALK2以及ALK3信号通路的抑制剂为LDN193189；
在扩增培养基中，所述Wnt信号通路的激动剂和所述GSK3抑 制剂均为CHIR99021；所述TGF-β信号通路的抑制剂为SB431542。
更具体的，在所述分化培养基中，所述bFGF的浓度为3-100ng/ml； 所述CHIR99021的浓度为0.3-30μM/l，所述SB431542的浓度为 2-50μM/l；所述Compound E的浓度为0.55-10μM/l；所述LDN193189 的浓度为0.1-10μM/l；在所述扩增培养基中，所述bFGF的浓度为 3-100ng/ml，所述CHIR99021的浓度为0.3-30μM/l；所述SB431542 的浓度为5-50μM/l；所述白血病抑制因子的浓度为50-5000U/l。
以下为本实施例的分化培养基和扩增培养基的具体实例，对于分 化培养基：
实例1：Neurobasal培养基、B-27添加剂、N-2添加剂、3ng/ml  bFGF、0.3μM/l CHIR99021、2μM/l SB431542、0.05μM/l Compound E 和0.1μM/l LDN193189；
实例2：Neurobasal培养基、B-27添加剂、N-2添加剂、100ng/ml  bFGF、30μM/l CHIR99021、50μM/l SB431542、10μM/l Compound E 和10μM/l LDN193189；
实例3：Neurobasal培养基、B-27添加剂、N-2添加剂、10ng/ml  bFGF、3μM/l CHIR99021、5μM/l SB431542、0.2μM/l Compound E 和0.1μM/l LDN193189。
对于扩增培养基：
实例1：Neurobasal培养基、B-27添加剂、N-2添加剂、3ng/ml  bFGF、0.3μM/l CHIR99021、5μM/l SB431542和50U/L白血病抑制 因子；
实例2：Neurobasal培养基、B-27添加剂、N-2添加剂、100ng/ml  bFGF、30μM/l CHIR99021、50μM/l SB431542和5000U/L白血病抑 制因子；
实例3：Neurobasal培养基、B-27添加剂、N-2添加剂、10ng/ml  bFGF、3μM/l CHIR99021、5μM/l SB431542和1000U/l白血病抑制因 子。
为了使得本发明实施例一的培养基得到更好的应用，更加有效应 用到神经上皮干细胞以及细胞系的建立中，本发明还在上述实施例一 的基础之上提供了实施例二，实施例二根据实施例一培养基建立神经 上皮干细胞的方法现做详细的阐述和解释，请参考图1-图5：
实施例二
一种利用权利要求1所述培养基获得神经上皮干细胞的方法，包 括以下步骤：
1、将多能干细胞经过胶原酶消化成细胞团块；
具体的，在该步骤中，将培养的饲养层或无饲养层培养的多能干 细胞经过胶原酶消化(5-40分钟)成小块的干细胞团块（50-100个细胞）。 另外，在本实施例中，多能干细胞，包括胚胎干细胞和诱导多能干细 胞。
2、将所述细胞团块悬浮在所述分化培养基中并进行悬浮培养， 使其分化为初级神经上皮干细胞；
具体的，将悬浮细胞团块在分化的培养基中，在低贴附的培养皿 中进行悬浮培养，进行连续6天的分化，每2天换液。在分化的第4 天开始，拟胚体出现两层样的神经上皮结构，请参考图1。将分化第 6天的神经上皮结构收集（静置5-10分钟，去掉上清液）。
在图1中，示出了多能干细胞定向诱导分化为高纯度的神经上皮 干细胞过程。其中，(A)为多能干细胞在分化培养基中分化第2天后 形成典型的拟胚体；(B-C)在分化的第5-6天，拟胚体形成神经上皮 结构，表达Nestin。(D-L)分别对分化后第2、5以及6天的拟胚体进 行冰冻切片，然后分别进行Oct4、Sox2、Nestin和Pax6的染色后显 示的阳性细胞染色结果。(M)为在拟胚体分化过程中Sox2、Oct4和 Pax6阳性细胞数量的定量变化(**P>0.01表示显著变化)。(N)为分 化的第6天和第12天拟胚体以及扩增的第6代的神经上皮干细胞样 品的RT-PCR（mRNA反转录PCR），表明多能干细胞很快失去了多 能性，转化为神经上皮干细胞。(O-T)为长期培养的神经上皮干细胞 表达干细胞的标志蛋白，如Sox2、Pax6、Nestin和N-cadherin，但不 表达放射胶质前体细胞的标志蛋白GFAP、神经元蛋白Tuj1(S)以及人 和猴发育的大脑皮层侧脑室外侧区域的前体细胞标志蛋白Tbr2(如图 1中T所示)。
3、将所述初级神经上皮干细胞使用所述扩增培养基进行培养， 获得能够稳定传代的神经上皮干细胞；
在该步骤中，通过扩增培养基将分化获得的初级神经上皮干细胞 培养之后，细胞长满之后则会得到神经上皮干细胞。
另外，为了获得能够使培养的细胞能自我组装成神经管的结构： 可进行如下操作：神经上皮干细胞需要在培养板上连续培养7-8天， 而不进行传代。这些传代的细胞保持了指数生长，甚至到50代后， 仍然保持旺盛的生长速度，其生长能力没有受到任何的影响。通过流 式细胞仪检测这些细胞，发现在长期的培养过程其稳定表达干细胞的 标志蛋白，如Nestin和Sox2。并且，这些长期传代培养的细胞保持 了分化为高纯度神经细胞的特性以及形成神经管结构的特性。请参考 图2：
在图2中，示出了神经上皮干细胞培养体系能够长期规模化培养 神经上皮干细胞，并保持干细胞自我组装为神经管的结构以及高纯度 神经元的产生。其中，(A)为第6天的拟胚体当培养在铺有laminin的 板上，3天后形成一个双层的神经上皮结构。(B)为低密度情况下分化 获得的神经上皮干细胞形成玫瑰花环（neural rosettes）结构。(C)为 在高密度的情况下神经上皮干细胞形成神经管结构。(D-E)长期培养 的神经上皮干细胞保持了神经管形成能力，并表达神经上皮干细胞标 志蛋白Nestin和ZO-1。(F-G)为分裂期标志蛋白phospho-vimentin (p-vimentin)染色显示分裂的细胞主要位于神经管的表面，而BrdU标 记的DNA合成期（S期）的细胞位于神经管的背面，显示存在细胞 分裂的核位移（interkinetic nuclear migration）。(H)为神经上皮干细胞 的生长曲线，表现为随着细胞的传代呈现指数生长。(I-J)为流式细胞 分选显示第18代和第36代的细胞表达相似的高比例Sox2和Nestin。 (K)为扩增96代后的神经上皮干细胞仍然具有正常的核型。(L-M)为 随着细胞的长期培养，保持了稳定高效的（>80%）Tuj1神经元分化 比率，而缺乏GFAP星型胶质细胞。
4、将所述神经上皮干细胞利用0.05%的胰酶进行消化传代，获 得稳定的神经上皮干细胞系；
优选的，在该步骤中，具体可以利用0.05%的胰酶进行消化传代， 传代比例为1：8-1：16。培养板事先用浓度5-50μg/ml的laminin进 行包被2小时以上，通过上述消化传代的操作，即可得到神经上皮干 细胞系。
此外，可通过单个神经上皮干细胞自我组装为神经管结构，具体 的，可以按照如下操作：将长期培养的单个神经上皮干细胞进行有限 稀释，分别培养在铺有laminin的96孔板的孔里，一个细胞/孔，培 养基采用上述的扩增培养基进行培养，每隔2-3天换液，直到第14-15 天，单个的细胞自我组装为一系列的神经管结构。这些单个细胞产生 的神经管结构表达Sox2、Nestin、Pax6、ZO-1和N-cadherin。BrdU 标记显示这些神经管结构具有很强的增值能力，并拥有细胞分裂时的 核位移（interkinetic nuclear migration）功能。如图3。
在图3中，示出了单个的神经上皮干细胞自我组装为神经管结构。 其中，(A)为单个神经上皮干细胞自我组装为神经管结构的连续克隆 示意图。(B)显示一个神经上皮干细胞在96孔板上。(C-D)为一个代 表性的单个神经上皮细胞所产生的神经管集落。D显示的是高倍镜下 的单个神经管结构。(E-H)为单个的神经上皮干细胞来源的神经管结 构表达神经上皮干细胞标志蛋白，如Pax6、Nestin、N-cadherin和 ZO-1。(I)BrdU标记显示单个细胞来源的神经管结构仍然保持很强的 增值能力。(J)显示出活细胞动态成像技术表示神经上皮干细胞具有相 似于体内的核位移（interkinetic nuclear migration），并表现出水平的 对称分裂。(K)为神经上皮干细胞长期培养过程中，存活的克隆比率 和形成神经管的极性克隆比例的变化，其结果显示随着神经上皮干细 胞传代时间的增加，细胞产生这两种类型集落的比例越高。(L)为在 连续的克隆过程中，接种细胞存活的细胞集落率以及存活的集落形成 神经管极性比率的比较。结果显示第一次克隆和第二次克隆之间没有 显著的差异(P>0.05)。
此外，在本实施例中，为了检测整个培养系统哪些关键性因素对 神经管的发育和产生所必需的，可利用单细胞形成神经管结构的特性， 通过在培养基中每次去掉一个成分进行一系列的单细胞培养实验，评 价哪些成分对神经上皮干细胞的自我更新和神经管形成所必需的。
研究发现去掉SB431542不会影响细胞形成神经管的能力。单独 去掉白血病抑制因子和bFGF将导致显著减少神经管的形成能力以及 细胞的增值能力，相反去掉CHIR99021没有一个细胞能够产生神经 管样的结构。如果采用SU5402抑制FGFR（成纤维生长因子受体） 信号通路，所有的细胞都失去了产生神经管的能力。因此，通过实验 说明，Wnt和FGFR信号是单个神经上皮干细胞形成神经管结构所必 需的。通过连续的二次克隆，我们发现白血病抑制因子是保持细胞连 续产生神经管结构所必需的。请参考图4：
在图4中，显示出了神经上皮干细胞自我组装为神经管结构以及 转化为放射胶质前体细胞的机制。(A)为单个神经上皮干细胞在不同 培养条件下，细胞存活以及形成极性神经管比率的比较。*P值＜0.05 表示显著差异,**P值＜0.01表示极显著差异。(B)为单个神经上皮干 细胞在不同实验条件下，细胞随着培养时间的延长，其增值能力差异 的比较。*P值＜0.05表示显著差异。(C-J)为Wnt信号通路的激活 是神经上皮干细胞形成神经管所必需的，而其失活将丢失神经管形成 的能力，并转化为放射胶质前体细胞。(K-N)显示了内源的FGFR信 号通路是神经管形成所必需的。SU5402是FGF受体的抑制剂。箭头 所指的是单个神经上皮干细胞在含有SU5402的神经上皮干细胞扩增 培养基中形成小的神经管结构。(O)为神经上皮干细胞自我组装为神 经管结构以及转化为放射胶质前体细胞的模型。
通过附图4可知，白血病抑制因子、FGF和Wnt信号通路在神 经管自我组装过程中的作用。外源bFGF和白血病抑制因子能促进神 经上皮干细胞的自我更新以及增加神经管结构的形成，而内源FGFR 和Wnt信号通路是神经管形成所必需的。另外，在Wnt信号通路失 活的情况下，神经上皮干细胞转化为放射胶质前体细胞，失去形成神 经管的能力。此外，早期转化的放射胶质前体细胞在Wnt信号通路 重新激活的情况下，能转化为神经上皮干细胞，而传代后的放射胶质 前体细胞则不能重新转化为神经上皮干细胞。
在步骤3之后，可选的，还可以利用神经上皮干细胞以及单个的 神经干细胞分化为高纯度的神经元。利用长期培养的神经上皮干细胞 以及单个细胞产生的稳定神经上皮干细胞系，在去掉bFGF、 CHIR99021、SB431542和LIF的条件下进行自发的分化，神经元的 分化培养基为Neurobasal培养基、B27、非必需氨基酸（含量可为1%） 和谷氨酰胺（浓度可为1mM）。分化细胞分别经过放射性胶质干细胞 （第0-3天）、中间神经前体细胞（第4-6天）的发育阶段，分化为 高纯度的神经元，这些神经元包括兴奋性谷氨酰胺能、抑制性γ-氨基 丁酸（GABA）以及大脑皮层的II-IV层、V-VI层的神经元、大脑皮 层的中间神经元，如图4中的D-J。
最后，将含有GFP的慢病毒加到神经上皮干细胞的扩增培养基 里面，感染培养的神经上皮干细胞，4小时后用PBS洗去病毒，换成 新鲜的培养基。将GFP标记的神经上皮干细胞以及单个GFP标记的 神经干细胞通过上述的单细胞扩增方法产生的细胞系通过立体定位 仪的方式，在麻醉的情况下，移植到食蟹猴的大脑脑部。3个月后， 将大脑进行固定，进行组织切片，发现移植细胞能够整合到食蟹猴的 大脑脑部，分化为神经元，这些神经元在3个月内，生长出长的轴突， 并广泛的分布于大脑皮层，部分的神经轴突往脑部的深层迁移。因此 该细胞具有重要的临床价值，可为将来的神经系统性疾病的干细胞治 疗提供重要的供体细胞来源。如图5中L-O。
在图5中，示出了利用单个神经上皮干细胞模拟人的神经管疾病， 并分化为大脑皮层神经细胞进行神经细胞替代治疗。(A-C)示出了单 个神经上皮干细胞转化为神经管能够模拟神经管发育缺陷疾病。其中， DHFR、SHMT1、MTRR、MTHFD1L、MTR以及CBS是叶酸代谢 中的关键酶(A)。这些酶高表达在神经上皮干细胞中(B)，并且叶酸 的浓度与神经管的形成呈正相关，而与集落的凋亡呈负相关(C)。(D-J) 为单个的神经上皮干细胞来源的稳定细胞系能分化产生大脑皮层神 经元。这些分化的神经元包括兴奋性谷氨酰胺能和抑制性γ-氨基丁酸 （GABA）神经元(D-G)。定量统计结果显示三株不同的单细胞系分 化的神经元比例没有显著的差异(H)。而且这些细胞系能够产生大脑 皮层II-IV层的Brn2神经元和V-VI层的Ctip2神经元，如I-J。(K-O) 示出单个神经上皮细胞来源的神经上皮干细胞移植到猴的视觉皮层 后，能够整合到大脑皮层(K)；这些移植的细胞整合到大脑皮层的最 外层(L-L”)和II-IV层(M-M”)，并分化为神经元。而且这些分化的神 经元生长出很长的轴突，这些轴突分布在大脑皮层的多个位点，并往 大脑皮层的深部延伸，如图5中O所述。
此外，通过本实施例的方法建立的神经上皮干细胞以及利用该神 经上皮细胞培养产生的细胞系；以及神经上皮干细胞和利用该神经上 皮细胞培养产生的细胞系在细胞替代治疗、疾病机制以及药物筛选中 的应用也属于本发明的保护范围。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明， 对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在 本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均 应包含在本发明的保护范围之内。

Claims (10)

  1. 一种建立神经上皮干细胞的培养基，其特征在于，包括：
    用于将多能干细胞诱导分化为初级神经上皮干细胞的分化 培养基以及用于使所述初级神经上皮干细胞扩增的扩增培养基；
    所述分化培养基包括：Neurobasal培养基、B-27添加剂、 N-2添加剂、bFGF、Wnt信号通路的激动剂、GSK3的抑制剂、 TGF-β信号通路的抑制剂、Notch信号通路抑制剂、ALK2以及 ALK3信号通路的抑制剂；
    所述扩增培养基包括：Neurobasal培养基、B-27添加剂、 N-2添加剂、bFGF、Wnt信号通路的激动剂、GSK3抑制剂、 TGF-β信号通路的抑制剂和白血病抑制因子。
  2. 根据权利要求1所述的建立神经上皮干细胞的培养基，其特征 在于：
    在所述分化培养基中，所述Wnt信号通路的激动剂和GSK3 抑制剂均为CHIR99021；所述TGF-β信号通路的抑制剂为 SB431542；所述Notch信号通路抑制剂为Compound E；所述 ALK2以及ALK3信号通路的抑制剂为LDN193189；
    和/或在所述扩增培养基中，所述Wnt信号通路的激动剂和 所述GSK3抑制剂均为CHIR99021；所述TGF-β信号通路的抑 制剂为SB431542。
  3. 根据权利要求2所述的建立神经上皮干细胞的培养基，其特征 在于，在所述分化培养基中，所述bFGF的浓度为3-100ng/ml； 所述CHIR99021的浓度为0.3-30μm/L，所述SB431542的浓度 为2-50μM/l；所述Compound E的浓度为0.05-10μM/l；所述 LDN193189的浓度为0.1-10μM/l；
    和/或在所述扩增培养基中，所述bFGF的浓度为 3-100ng/ml，所述CHIR99021的浓度为0.3-30μM/l；所述 SB431542的浓度为5-50μM/l；所述白血病抑制因子的浓度为 50-5000U/l。
  4. 一种利用权利要求1所述的培养基获得神经上皮干细胞的方法， 其特征在于，包括以下步骤：
    将多能干细胞经过胶原酶消化成细胞团块；
    将所述细胞团块悬浮在所述分化培养基中并进行悬浮培养， 使其分化为初级神经上皮干细胞；
    将所述初级神经上皮干细胞使用所述扩增培养基进行培养， 获得能够稳定传代的神经上皮干细胞。
  5. 根据权利要求4所述的方法，其特征在于；在所述步骤将所述 初级神经上皮干细胞使用所述扩增培养基进行培养，获得能够 稳定传代的神经上皮干细胞之后，还包括：
    将所述神经上皮干细胞进行稀释，得到单个神经上皮干细 胞；
    将所述单个神经上皮干细胞利用所述扩增培养基培养 14-15天，得到该单个神经上皮干细胞自我组装成的神经管。
  6. 根据权利要求4所述的方法，其特征在于，在所述步骤将所述 初级神经上皮干细胞使用所述扩增培养基进行培养，获得能够 稳定传代的神经上皮干细胞之后，还包括：
    将所述神经上皮干细胞在含有Neurobasal培养基、B27、 非必需氨基酸以及谷氨酰胺的神经元分化培养基上培养并使其 分化，得到纯度为40%-100%的神经元。
  7. 根据权利要求4所述的方法，其特征在于，在所述将多能干细 胞经过胶原酶消化成细胞团块的步骤中：
    所述消化时间为5-40分钟。
  8. 根据权利要求4所述的方法，其特征在于，在所述将所述初级 神经上皮干细胞使用所述扩增培养基进行培养，获得能够稳定 传代的神经上皮干细胞的步骤之后，还包括：
    将所述神经上皮干细胞利用0.05%的胰酶进行消化传代， 获得稳定的神经上皮干细胞系。
  9. 一种根据权利要求4-8任一项所述的方法建立的神经上皮干细 胞以及利用该神经上皮细胞培养产生的细胞系。
  10. 一种根据权利要求9所述的神经上皮干细胞以及利用该神经上 皮细胞培养产生的细胞系在细胞替代治疗、疾病机制以及药物 筛选中的应用。
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