Generation of neural precursor cells from embryonic stem cells or induced pluripotent stem cells

Abstract

The present application provides methods, cell culture media and combinations thereof for generating neural precursor cells (NPCs), especially human neural precursor cells (hNPCs), from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), wherein the NPCs are particularly suitable for preclinical and clinical applications.

Description

Technical Field

[0001] The present invention relates to methods, cell culture media and combinations thereof for generating neural precursor cells (NPCs), in particular human neural precursor cells (hNPCs), from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), wherein the NPCs are particularly suitable for preclinical and clinical use. Background Art

[0002] Neural progenitor cells are particularly valuable in cell therapy because they can not only self-renew and differentiate into all types of neural cells, but also migrate and integrate into damaged parts of the central nervous system. At the same time, these transplanted NPCs can secrete a variety of neuroprotective and angiogenic cytokines and microRNAs to enhance the autonomous healing process. Therefore, cell replacement therapy using multipotent NPCs to repair brain and spinal cord injuries is very promising.

[0003] Since 2008 (Dimos et al., 2008), the method of inducing differentiation of human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs) into adult neural cells in vitro, and its use in various aspects such as nervous system diseases, drug screening, drug neurotoxicity testing, transplantation research, etc., has become a hot topic in scientific research and drug development. Neural stem cells or NPCs obtained by the previously existing methods of inducing neural differentiation can only be further differentiated into specific subtypes of neural cells, whether in vitro or in vivo, which are usually difficult to have relatively mature functions, and there are also problems such as short survival time and limited yield.

[0004] In 2016, Xu et al. (Xu et al., Science Translational Medicine, 2016) proposed a new neural differentiation method that can be used to differentiate high-purity forkhead box G1 positive (FOXG1) from pluripotent stem cells in batches. + ) NPCs, the differentiated NPCs can further differentiate into neural cells that correctly correspond to various cell types of different cerebral cortex, including various excitatory and inhibitory neurons and astrocytes, and have mature electrophysiological functions and longer survival in vitro. However, the method published in this article uses animal-derived reagents and components of uncertain composition. Therefore, it is very necessary to develop a stable method for generating NPCs with stable quality and suitable for clinical application from pluripotent stem cells. Summary of the invention

[0005] The inventors of the present invention have established a set of methodologies capable of generating NPCs suitable for clinical and preclinical applications from ESCs or iPSCs via directed differentiation, thereby meeting the above-mentioned needs and completing the present invention.

[0006] Therefore, in a first aspect, the present application relates to a method for generating NPCs from ESCs or iPSCs, comprising:

[0007] (1) Cultivating ESCs or iPSCs in human pluripotent stem cell culture medium (hPSC culture medium) to form embryoid bodies (EBs);

[0008] (2) culturing the EBs formed by step (1) in an EB medium comprising a basal medium and an additive;

[0009] (3) culturing the EBs after step (2) on a culture surface coated with an extracellular matrix (ECM) using a neural induction medium (NIM) to form rosette neural aggregates (RONAs), wherein the NIM comprises a basal medium and additives;

[0010] (4) culturing the RONAs formed by step (3) to form neurospheres; and

[0011] (5) Dispersing the neurospheres into single cells and culturing them on a culture surface coated with ECM using NPC medium to form a monolayer of NPCs,

[0012] The hPSC culture medium, and various basic culture media and additives contained in the EB culture medium, NIM culture medium and NPC culture medium are of clinical grade.

[0013] In a further embodiment, one or more, preferably all, of the various basal media and additives contained in the EB medium, NIM medium, and NPC medium are of GMP grade, cGMP grade, or CTS grade. TM More preferably, one or more, more preferably two, three or four, of the hPSC culture medium, EB culture medium, NIM and NPC culture medium are of clinical grade, preferably GMP grade, cGMP grade or CTS grade. TM class.

[0014] In specific embodiments, the culture surface is a culture plate or a culture flask.

[0015] In one embodiment, the hPSC culture medium is clinical grade, preferably GMP grade, cGMP grade or CTS grade. TM Specifically, the hPSC culture medium is selected from the following: hPSC XF Medium, CTS TM Essential 8 Medium, Basic03 culture medium, StemMACS TM iPS-Brew medium, ACF medium, TeSR TM -AOF medium and TeSR2 medium. In a preferred embodiment, the hPSC medium is hPSC XF Medium.

[0016] In one embodiment, the hPSC culture medium is supplemented with a ROCK inhibitor. In a preferred embodiment, the ROCK inhibitor is clinical grade, preferably GMP grade, cGMP grade or CTS grade. TM class.

[0017] In one embodiment, the EB culture medium comprises:

[0018] (i) a basal medium selected from a) to c):

[0019] a) Single KnockOut TM DMEM / F12 medium,

[0020] b) DMEM / F12 medium and Neurobasal TM Combination of culture media,

[0021] c) KnockOut TM DMEM / F12 medium and Neurobasal TM Combination of culture media;

[0022] (ii) an additive comprising or consisting of d) or e):

[0023] d) N-2 Supplement and GlutaMAX TM -I additives;

[0024] e) N-2 additives, GlutaMAX TM -I Supplement and B-27 without Vitamin A TM Additive (B-27 TM Supplement, minus vitamin A); and

[0025] (iii) optional inhibitors.

[0026] In a further embodiment, the EB culture medium comprises:

[0027] (i) a basal medium selected from a) to c):

[0028] a) Single CTS TM KnockOut TM DMEM / F12 medium,

[0029] b) cGMP grade DMEM / F12 medium and CTS TM Neurobasal TM Combination of culture media,

[0030] c) CTS TM KnockOut TM DMEM / F12 medium and CTS TM Neurobasal TM Combination of culture media;

[0031] (ii) an additive comprising or consisting of d) or e):

[0032] d) CTS TM N-2 Additives and CTS TM GlutaMAX TM -I additives; and

[0033] e) CTS TM N-2 additive, CTS TM GlutaMAX TM -I additives, and cGMP grade or CTS TM XenoFree B-27 TM additive;

[0034] (iii) optional inhibitors.

[0035] In a preferred embodiment, when the basal medium of the EB culture medium is a), the additive comprises e) or consists of e). In a specific embodiment, the additive of the EB culture medium is d) or e).

[0036] In a preferred embodiment, the EB culture medium comprises an inhibitor. More specifically, the inhibitor comprises a BMP inhibitor, an AMPK inhibitor and an ALK inhibitor, or is composed of a BMP inhibitor, an AMPK inhibitor and an ALK inhibitor. In a more specific embodiment, the inhibitor comprises one or more of Noggin, SB431542, LDN-193189, DMH-1 and Dorsomorphin, or is composed of one or more of Noggin, SB431542, LDN-193189, DMH-1 and Dorsomorphin. Preferably, the EB culture medium comprises SB431542 and any one or more of Noggin, LDN-193189, DMH-1 and Dorsomorphin, such as a combination of one or two of Noggin, LDN-193189, DMH-1 and Dorsomorphin. In a specific embodiment, the EB culture medium comprises Noggin, Dorsomorphin and SB431542. In a preferred embodiment, one or more, preferably all, of the inhibitors are of clinical grade, preferably GMP grade, cGMP grade or CTS grade. TM class.

[0037] In one embodiment, the NIM comprises:

[0038] (i) a basal medium selected from a) to c):

[0039] a) Single KnockOut TM DMEM / F12 medium,

[0040] b) DMEM / F12 medium and Neurobasal TM Combination of culture media,

[0041] c) KnockOut TM DMEM / F12 medium and Neurobasal TM Combinations of culture media; and

[0042] (ii) an additive comprising or consisting of d) or e):

[0043] d) N-2 Supplement and GlutaMAX TM -I additives;

[0044] e) N-2 additives, GlutaMAX TM -I Supplement and B-27 without Vitamin A TM additive.

[0045] In one embodiment, the NIM comprises:

[0046] (i) a basal medium selected from a) to c):

[0047] a) Single CTS TM KnockOut TM DMEM / F12 medium,

[0048] b) cGMP grade DMEM / F12 medium and CTS TM Neurobasal TM Combination of culture media,

[0049] c) CTS TM KnockOut TM DMEM / F12 medium and CTS TM Neurobasal TM Combinations of culture media; and

[0050] (ii) an additive comprising or consisting of d) or e):

[0051] d) CTS TM N-2 Additives and CTS TM GlutaMAX TM -I additives; and

[0052] e) CTS TM N-2 additive, CTS TM GlutaMAX TM -I additives, and cGMP grade or CTS TM Grade Vitamin A-Free Xeno-Free B-27 TM additive.

[0053] In a preferred embodiment, when the basal medium of the NIM medium is a), the additive comprises e) or consists of e). In a specific embodiment, the additive of the NIM medium is d) or e).

[0054] The step (4) of culturing RONA into neurospheres uses the same culture medium as the previous step or the next step. In one embodiment, the culture medium used in the step (4) of culturing RONA into neurospheres is the same as that in the previous step (step (3)), specifically NIM. In another embodiment, the culture medium used in the step (4) of culturing RONA into neurospheres is the same as that in the next step (step (5)), specifically NPC culture medium is used, and the NPC culture medium comprises a basal culture medium and an additive.

[0055] In one embodiment, the NPC culture medium is supplemented with GlutaMAXTM -I Supplement and B-27 without Vitamin A TM Additives for Neurobasal TM In a further embodiment, the NPC culture medium is supplemented with CTS TM GlutaMAX TM -I additives and cGMP grade or CTS TM Grade Vitamin A-Free Xeno-Free B-27 TM CTS of Additives TM Neurobasal TM In a preferred embodiment, the NPC culture medium further comprises brain-derived neurotrophic factor (BDNF), and / or glial cell-derived neurotrophic factor (GDNF), and / or L-ascorbic acid, and / or N 6 ,O 2’ -Dibutyryladenosine 3',5' cyclic monophosphate sodium salt (DB-cAMP). In more specific embodiments, the BDNF is animal-free recombinant BDNF or GMP-grade recombinant BDNF, and / or the GDNF is animal-free recombinant GDNF or GMP-grade recombinant GDNF. In preferred embodiments, one or more of the neurotrophic factors are GMP-grade, cGMP-grade or CTS-grade. TM More preferably, each of the neurotrophic factors is of GMP grade, cGMP grade or CTS grade. TM class.

[0056] In one embodiment, the neurospheres are dispersed using digestive enzymes in step (5). In another embodiment, the neurospheres are dispersed by mechanical means in step (5).

[0057] In one embodiment, prior to step (1), the ESCs or iPSCs are maintained in culture and expanded to 80% to 90% confluence, preferably cultured on a culture surface coated with laminin. The culture surface may be a culture plate. The laminin used for coating is preferably clinical grade.

[0058] In one embodiment, wherein in step (1), ESCs or iPSCs are dispersed into single cells or cell aggregates, for example by digestive enzymes or by mechanical means, and then inoculated into hPSC culture medium to induce EBs formation. Preferably, digestive enzymes are used for digestion to obtain better and more uniformly dispersed cells. In one embodiment, the stem cells are inoculated into ultra-low adsorption 96-well culture plates at a density of 5,000 to 40,000 cells / well, preferably about 10,000 to 35,000 cells / well, more preferably about 20,000 to 30,000 cells / well, and most preferably 30,000 cells / well, and are cultured using hPSC culture medium. In one embodiment, the stem cells are inoculated into low adsorption culture plates in the form of clonal colonies and cultured using the hPSC culture medium.

[0059] In one embodiment, the digestive enzyme used in step (1) or step (5) is CTS TM TrypLE TM In one embodiment, the digestive enzyme is clinical grade, GMP grade, cGMP grade or CTS TM class.

[0060] In a specific embodiment, the culture in step (2) is suspension culture. In a specific embodiment, the culture in step (3) is adherent culture.

[0061] In another embodiment, more than one, such as two, three or more EB culture media of the present invention are used in step (2). In another embodiment, more than one, such as two, three or more NIMs of the present invention are used in step (3).

[0062] In a second aspect, the present invention relates to NPCs produced by the method of the first aspect, or cells derived from NPCs produced by the method of the first aspect. For example, the cells derived from NPCs can be neurons, astrocytes, oligodendrocyte precursor cells or oligodendrocytes.

[0063] In one embodiment, the majority of NPCs generated by the method of the first aspect are FOXG1 + For example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the generated NPCs are FOXG1 + NPCs.

[0064] In a preferred embodiment, the NPCs or self-derived cells produced by the method of the first aspect are suitable for preclinical and clinical applications.

[0065] In a third aspect, the present invention relates to use of the NPCs or cells derived therefrom according to the second aspect in drug development or clinical applications.

[0066] In a fourth aspect, the present invention relates to a method for preparing neurons, astrocytes, oligodendrocytes, oligodendrocytes, or a mixed cell population comprising any one or more thereof from NPCs produced by the method of the first aspect. In one embodiment, the method comprises further differentiating the NPCs. In another preferred embodiment, the method uses clinical grade, GMP grade, cGMP grade or CTS grade TM grade culture medium.

[0067] In a fifth aspect, the present invention relates to neurons, astrocytes, astrocytes, oligodendrocytes or oligodendrocytes differentiated from NPCs produced by the method of the first aspect. For example, neurons, astrocytes, astrocytes, oligodendrocytes or oligodendrocytes obtained by the method of the fourth aspect. Preferably, the neurons, astrocytes, astrocytes, oligodendrocytes or oligodendrocytes are suitable for preclinical and clinical applications.

[0068] As can be seen from the foregoing, the EB medium and NIM can share the same important components that contribute to the success of the present invention, including basal medium and additives. Therefore, in a sixth aspect, the present invention relates to the use of a combination of basal medium and additives in the preparation of EB medium and / or NIM in the process of generating NPCs by differentiation from ESCs or iPSCs, wherein the basal medium is DMEM / F12 or KnockOut TM DMEM / F12, and the additives were N-2 supplement and GlutaMAX TM -I additives, and wherein the basal medium and additives are clinical grade. Preferably, one or more of the basal medium and additives are GMP grade, cGMP grade or CTS grade. TM In one embodiment, the basal medium further comprises clinical grade Neurobasal TM Culture medium, preferably CTS TM Neurobasal TM In another embodiment, the additive further comprises clinical grade vitamin A-free B-27 TM Additives, preferably cGMP grade or CTS TM Xeno-Free B-27 Without Vitamin A TM additive. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] In order to facilitate a better understanding of the features and advantages of the present invention, the following detailed description of the invention and the accompanying drawings are provided. However, those skilled in the art should understand that these contents are only for the purpose of illustrating the present invention, rather than limiting the present invention. The scope of the present invention shall be subject to the claims.

[0070] Figure 1 A flow chart illustrating the generation of human neural progenitor cells (hNPCs) from human pluripotent stem cells (hPSCs), showing the key steps, with bright-field micrographs showing the appearance of the cell culture at the end of each step.

[0071] Figure 2 Bright field images of clinical-grade hNPCs differentiated from hESCs (Example 1) are shown.

[0072] Figure 3 The immunofluorescence staining results of clinical-grade hNPCs obtained in Example 1 of the present invention are shown. The term "DAPI" as used herein means 4',6-diamidino-2-phenylindole.

[0073] Figure 4 The flow cytometric detection results of clinical-grade hNPCs obtained in Example 1 of the present invention are shown.

[0074] Figure 5A -D shows the characterization results of clinical-grade human neural cells, which are obtained by in vitro differentiation of hNPCs in Example 1 of the present invention. Figure 5A and Figure 5B The cerebral cortical neuron markers (BRN2, CTIP2, TBR1 and SATB2) of the clinical-grade hNPCs obtained by expansion and culture in Example 1 of the present invention after 2 months of in vitro differentiation were shown; Figure 5A ) and synaptic protein markers (Synapsin and PSD95; Figure 5B ) immunofluorescence staining results; Figure 5C The results of electrophysiological activity testing of clinical-grade human neural cells on days 6 and 13 of in vitro differentiation are shown; Figure 5D The immunofluorescence staining results of glial cell markers of clinical-grade hNPCs obtained by expansion and culture in Example 1 of the present invention after 3 months of in vitro differentiation are shown, wherein the cell nucleus is marked by DAPI, the astrocytes are marked by GFAP, and the oligodendrocyte precursor cells are marked by OLIG2.

[0075] Figure 6 This is the growth curve of clinical-grade hNPCs obtained by expansion and culture in Example 1 of the present invention.

[0076] Figure 7 This is a bright field image of clinical-grade hNPCs differentiated from hiPSCs in Example 4 of the present invention.

[0077] Figure 8 This is the immunofluorescence staining result of clinical-grade hNPCs expanded and cultured in Example 4 of the present invention.

[0078] Fig. 9 This is the flow cytometry test result of the clinical-grade hNPCs amplified and cultured in Example 4 of the present invention.

[0079] Fig. 10A -D shows the characterization results of clinical-grade human neural cells, which are obtained by in vitro differentiation of hNPCs of Example 4 of the present invention. Fig. 10A and Fig. 10B The cerebral cortical neuron markers (BRN2, CTIP2, TBR1 and SATB2) of the clinical-grade hNPCs obtained by expansion and culture in Example 4 of the present invention after 2 months of in vitro differentiation were shown; Fig. 10A ) and synaptic protein markers (Synapsin and PSD95; Fig. 10B ) immunofluorescence staining results; Fig. 10C The results of electrophysiological activity testing of clinical-grade human neural cells on days 6 and 14 of in vitro differentiation are shown; Fig. 10D The immunofluorescence staining results of glial cell markers of clinical-grade hNPCs obtained by expansion and culture in Example 4 of the present invention after 3 months of in vitro differentiation are shown, wherein the cell nuclei are marked by DAPI, astrocytes are marked by GFAP, and oligodendrocyte precursor cells are marked by OLIG2.

[0080] Fig.11A -E shows the results of the cell differentiation method using different media combinations described in Example 7 and Table 2. Fig.11A , 11B , 11C, 11D, and 11E show the results of combinations 1, 4, 5, 8, and 10, respectively.

[0081] Fig.12 Bright field images of EBs formed by the method of Example 8 are shown.

[0082] Fig.13 This is the immunofluorescence staining result of hNPCs amplified and cultured in Example 9 of the present invention.

[0083] Fig.14A -D shows the characterization results of clinical-grade human neural cells, which are obtained by in vitro differentiation of hNPCs of Example 9 of the present invention. Fig.14A and Fig. 14BThe cerebral cortical neuron markers (BRN2, CTIP2, TBR1 and SATB2) of the clinical-grade hNPCs obtained by expansion and culture in Example 9 of the present invention after 2 months of in vitro differentiation were shown; Fig.14A ) and synaptic protein markers (Synapsin and PSD95; Fig. 14B ) were obtained from the immunofluorescence staining of . Fig. 14C Shown are the results of electrophysiological activity testing of clinical-grade human neural cells on days 7 and 14 of in vitro differentiation. Fig.14D The immunofluorescence staining results of glial cell markers of clinical-grade hNPCs obtained by expansion and culture in Example 9 of the present invention after 3 months of in vitro differentiation are shown, wherein the cell nuclei are marked by DAPI, neurons are marked by Map2, astrocytes are marked by GFAP, and oligodendrocyte precursor cells are marked by OLIG2. DETAILED DESCRIPTION OF THE INVENTION

[0084] Unless otherwise specified in the present invention, the meanings of all technical and scientific terms used herein are to be interpreted according to the common understanding by one of ordinary skill in the art to which the present invention belongs.

[0085] "Or" has the same meaning as "and / or" herein unless explicitly stated.

[0086] In the context of the present invention, unless otherwise stated, "comprising", "including" and "containing" should be understood to mean including the listed elements, such as a component, a feature, a step or a group thereof, but not excluding any other elements, such as other components, properties and steps. When used herein, the term "comprising" or any variation thereof can be replaced with "containing", "including" or "having" or synonymous variations. In certain embodiments, "comprising" also includes the situation of "consisting of..."

[0087] Clinical Grade

[0088] Without limiting the use of reagents, culture media and cell-derived materials from other sources, whether known or unknown, in the methods of the present application, with respect to materials used to produce hNPCs, in particular one or more materials constituting the culture medium of the present application, as well as matrix and enzymes, the term "clinical grade" indicates that the reagents, culture media or cell-derived materials are suitable for clinical applications themselves and / or can be used to direct the differentiation of stem cells, in particular embryonic stem cells or induced pluripotent stem cells, into safe and stable cells suitable for clinical applications, in particular hNPCs.

[0089] More preferably, the reagents, culture media or cell-derived materials used to produce the hNPCs of the present invention, in particular one or more materials constituting the culture media of the present application, are of GMP (Good Manufacturing Practice) grade or cGMP (Current Good Manufacturing Practice) grade, which means that the reagents, culture media or cell-derived materials have certified GMP or cGMP quality and are produced under GMP or cGMP standards, which are established by authoritative organizations such as the World Health Organization, the Ministry of Health of the People's Republic of China, the U.S. Food and Drug Administration or the European Medicines Agency. "GMP" or "cGMP" means the (current) standards that manufacturers should follow to ensure that the production process of their products is properly monitored and controlled, and that the products they provide to end users are always highly safe.

[0090] The term "CTS" or "CTS TM " is the abbreviation of cell therapy systems. This term is used by Thermo Fisher to indicate that a product produced for cell therapy is of high quality and meets cGMP standards.

[0091] In the context of this application, the phrase "clinically applicable" or "suitable for clinical application" in relation to NPCs means that the NPCs at least meet the standards required by specific regulations related to clinical and preclinical practice, such as meeting good manufacturing standards (GMP). Specifically, the NPCs differentiated by the method of the present invention have properties that make them suitable for clinical application.

[0092] Cell types at different stages

[0093] The term "neural precursor cell" or "NPCs" as used herein refers to a cell type that can differentiate into cells in a neural lineage, including but not limited to neurons, glial cell precursors and glial cells, such as astrocyte precursor cells, astrocytes, oligodendrocyte precursors and oligodendrocytes. Precursor cells are stem cell-like cells, but their replication and proliferation abilities are weaker than stem cells. However, compared with fully differentiated cells, precursor cells still have the ability to differentiate into different cell types. Therefore, precursor cells can be used as safer seed cells for cell replacement therapy. Whether a cell (particularly NPCs) successfully differentiates to obtain a specific cell type can be determined by morphological observation, cell-specific biomarker detection, and in vitro differentiation to obtain a specific neural cell type with a specific biomarker and electrophysiological activity. For example, NPCs produced by the method of the present invention can show a typical neural precursor cell morphology, including a relatively homogeneous columnar cell body and a rosette-like radial cell arrangement.

[0094] The abbreviation "FOXG1" or "Foxg1" as used herein stands for "forkhead box G1", which is one of the earliest transcription factors expressed during human brain development, inducing the development of the telencephalon into several key structures, including the cerebral cortex. The cerebral cortex is derived from an organ primordium that expresses the forebrain forkhead box G1 (FOXG1). Therefore, FOXG1 can serve as a marker for precursor cells that will eventually differentiate into forebrain cells. In one embodiment of the present invention, NPCs produced by the method of the present invention express FOXG1.

[0095] The term "embryonic stem cells" or "ESCs" as used herein refers to pluripotent stem cells derived from embryos. The term "induced pluripotent stem cells" or "iPSCs" as used herein refers to pluripotent stem cells produced by reprogramming differentiated somatic cells. The method of the present invention can use ESCs or iPSCs as the starting material for directed differentiation. In a specific embodiment of the present invention, ESCs or iPSCs are of mammalian origin, particularly human origin. The present invention does not limit the source of stem cells including ESCs and iPSCs, as long as they meet the requirements of clinical applications. The cells can be obtained directly from commercial sources or can be prepared, for example, by reprogramming somatic cells into iPSCs.

[0096] The term "embryoid body" or "EBs" as used herein refers to cell aggregates grown from ESCs and iPSCs in three-dimensional culture. EBs can be formed by suspension culture. However, it is difficult to obtain EBs of uniform size and shape by traditional culturing of EBs.

[0097] In the context of the present invention, the terms "neural aggregates derived from rosette-like neural stem cells", "rosette-like neural aggregates" or "RONAs" are used interchangeably herein and refer to aggregates of neural stem cells derived from ESCs or iPSCs, which are spontaneously organized by neural stem cells to form highly compact three-dimensional columnar neural aggregates. In the method of the present invention, specifically, the rosette-like neural aggregates formed in step (3) are positive for FOXG1 and Nestin in immunostaining. In the method of the present application, RONAs are usually formed 8 days after initial differentiation, that is, when EBs are transferred to the coated culture surface (such as a culture plate) of step (3) of the present invention and cultured for about 1 day.

[0098] In the context of the present application, the term "neurosphere" as used herein refers to spherical aggregates formed by suspension culture of neural fate cells separated from RONAs. Neurospheres are a heterogeneous population composed of different types of neural cells, including neural stem cells, neural precursor cells and some differentiated neural cells. In the method of the present application, neurospheres are mainly composed of NPCs, which can be dispersed or digested into single cells and then inoculated onto a culture surface coated with matrix gel, such as inoculated into a cell culture plate, to form a high-purity monolayer of NPCs. In the method of the present application, neurospheres are usually formed 21 days after the start of differentiation, that is, after RONAs are cultured in the culture medium of step (4) of the present invention for about 1 day. The process of culturing RONAs into neurospheres can use the same culture medium as the previous culture step or the next culture step, such as NIM used to culture RONAs or NPC culture medium used to form a monolayer of NPCs.

[0099] In the present invention, mechanical means or enzymes (such as enzymes capable of separating cell matrix) can be used to separate cells from a culture surface (such as a culture plate) or disperse neurospheres. In a preferred embodiment, compared with mechanical means, the use of digestive enzymes for such separation or dispersion can disperse cells that were originally aggregated together into more uniform single discrete cells. Specifically, digestive enzymes can be used for treatment in different steps of the method to achieve different purposes. For example, in step (5) of the present invention, digestive enzymes are used to disperse neurospheres into single cells. In addition, digestive enzymes can also be used for the following purposes: (a) before inoculating cells in step (1) of the method, stem cells are dispersed and suspended, and / or (b) NPCs are dispersed during the passage process after step (5) of the method. The digestive enzymes used in the aforementioned steps may be the same or different. In a specific embodiment, the enzymes used in the method of the present invention, especially the enzymes used in step (1), can digest cell colonies into discrete single cells, which is different from enzymes that only peel cell colonies from a culture surface such as a culture plate while the colonies are still connected to each other. Exemplary enzymes can be Accutase, Dispase, Versene (EDTA) or TrypLE. In a preferred embodiment, the enzyme is CTS TM TrypLE TM Select enzyme, which can be used to digest stem cells (ESCs or iPSCs), neurospheres and NPCs. In another embodiment, the enzyme used to disperse the neurospheres in step (5) is CTS TM TrypLE TM Preferably, the digestive enzyme or cell separation enzyme solution suitable for digesting or separating cells in the method of the present invention is clinical grade, GMP grade, cGMP grade or CTS grade. TMThe digestive enzyme is suitable for preparing NPCs for clinical application. The enzyme can be used in the dosage recommended by its manufacturer.

[0100] For the culture of iPSCs / ESCs, RONAs and monolayer NPCs, the culture surface such as a culture plate is coated with an extracellular matrix (ECM) to provide support for cell attachment and growth. ECM is an extracellular matrix and is mainly composed of proteins such as collagen, elastin and laminin. ECM is widely used in the culture of mammalian cells and is also known to those skilled in the art. Non-limiting examples of ECM that can be used to coat solid supports or culture surfaces such as culture plates include Matrigel TM , laminin, poly-lysine, a combination of poly-ornithine (PO) / fibronectin (FN) / laminin (lam), fibronectin (FN), etc. In a preferred embodiment, the culture surface such as a culture plate used to culture RONAs is coated with Matrigel TM In a more preferred embodiment, the culture surface such as a culture plate used to culture RONAs is coated with Laminin-521 obtained from BioLamina, in particular MX521 or CT521, which is clinical grade or GMP grade.

[0101] Whether a cell has differentiated or transformed into a specific type of cell can be determined by immunostaining for protein markers that are specifically expressed in specific cell types. Commonly used markers in neuromics are well known to those skilled in the art. For example, microtubule-associated protein 2 (Map2) a, b, and c subtypes are only expressed in neurons, specifically in the perikarya and dendrites.

[0102] The present application also relates to a method for further producing differentiated cells from NPCs produced by the method of the present invention, wherein the differentiated cells include but are not limited to neurons and glial cells, such as astrocytes and oligodendrocytes, and the method is performed by, for example, culturing the NPCs in a differentiation medium. The differentiation conditions including the differentiation medium can be determined by a person skilled in the art according to the cell type desired to be cultured. Preferably, the differentiation medium is clinical grade, GMP grade, cGMP grade or CTS grade. TM level, thus making the cells differentiated from NPCs suitable for preclinical and clinical applications.

[0103] In a specific embodiment, the NPCs produced by the method of the present invention are cultured in a neural differentiation medium to produce neurons. The NPC medium of the present invention, such as CTS-NPC medium, can be used as a neural differentiation medium, which reduces the complexity of the entire process of differentiating neurons from stem cells and can produce neurons suitable for preclinical and clinical applications.

[0104] Culture medium

[0105] The composition of the culture medium used in each step of the present application is crucial for the successful differentiation of NPCs. Any culture medium and components contained therein in the present application should be clinical grade, preferably GMP grade, cGMP grade or CTS grade. TM level, or can be used to differentiate into NPCs suitable for clinical applications.

[0106] The culture medium is a mixture that contains various nutrients required for the growth of a certain type of cell. The culture medium can be prepared by adding additives to the basal culture medium. Additives in a broad sense refer to additional components required for cell culture but not included in the basal culture medium, including proteins, lipids, amino acids, vitamins, hormones, cytokines, growth factors, etc. However, in this article, the term "additive" does not include inhibitors added separately to hPSC culture medium or EB culture medium, nor does it include L-ascorbic acid, DB-cAMP, and neurotrophic factors added separately to NPC culture medium. Any basal culture medium and additives used in the method of the present invention are clinical grade, preferably GMP grade, cGMP grade or CTS grade. TM class.

[0107] In the context of the present application, hPSC culture medium is the culture medium for culturing ESCs or iPSCs to form EBs in step (1) of the present method. The hPSC culture medium suitable for the present method can be selected from the following group: XF medium, CTS TM Essential 8 Medium, Basic03 culture medium, StemMACS TM iPS-Brew medium, ACF medium, TeSR TM -AOF medium and TeSR2 medium.

[0108] In a preferred embodiment, a culture medium supplemented with a ROCK inhibitor can be used in step (1) of the method of the present invention. ROCK inhibitors are a class of protein kinase inhibitors that inhibit the activity of rho-associated protein kinase (ROCK) belonging to the serine-threonine protein kinase family and prevent cells from apoptosis after digestion or resuscitation. ROCK is involved in regulating the shape and movement of cells by acting on the cytoskeleton, and also regulates the immortalization and differentiation of cells. Exemplary ROCK inhibitors include but are not limited to Y-27632. In order to differentiate NPCs suitable for clinical applications, clinical grade, preferably GMP grade, cGMP grade or CTS grade, is used in the method of the present application. TM These ROCK inhibitors can be obtained commercially. ROCK inhibitors can be added to hPSC culture medium at a concentration of about 10 μM.

[0109] In the context of the present application, EB culture medium is a culture medium used to culture EBs and induce neural differentiation. Specifically, EB culture medium refers to the culture medium used in step (2) of the method of the present invention.

[0110] In a preferred embodiment, the EB culture medium comprises an inhibitor. More specifically, the inhibitor comprises a BMP inhibitor, an AMPK inhibitor and an ALK inhibitor, or consists of a BMP inhibitor, an AMPK inhibitor and an ALK inhibitor. In one embodiment, the EB culture medium comprises one or more inhibitors selected from the group consisting of Noggin, Dorsomorphin, DMH-1, LDN-193189 and SB431542. Preferably, the EB culture medium comprises a combination of SB431542 and one or more of Noggin, LDN-193189, DMH-1 and Dorsomorphin, for example, a combination of SB431542 and one or two. More specifically, the EB culture medium comprises a combination of Noggin, SB431542 and Dorsomorphin. The concentration of Noggin in the EB culture medium may be 25-100 ng / mL, preferably 40-60 ng / mL, and most preferably 50 ng / mL. The concentration of Dorsomorphin in the EB medium may be 0.5-2 μM, preferably 0.75-1.5 μM, and most preferably 1 μM. The concentration of SB431542 in the EB medium may be 5-15 μM, preferably 7-13 μM, more preferably 8-12 μM, and most preferably 10 μM.

[0111] In a specific embodiment, the basal medium and additives of the EB medium can be selected from Table 1. In a more specific embodiment, the EB medium has a combination of specific basal medium and additives listed in Table 1 below.

[0112] In the context of this application, NIM is the culture medium used to induce the formation of RONAs.

[0113] When the EB medium or the NIM basal medium is prepared by mixing two basal mediums, the mixing volume ratio of the two mediums is about 1:1.

[0114] In one embodiment, the NIM and EB culture media are composed of the same basic culture media and additives, and the only difference is that the EB culture media is supplemented with inhibitors. Using NIM and EB culture media with exactly the same basic components can make the preparation of the culture media more efficient.

[0115] In a specific embodiment, the basal medium and additives of the NIM can be selected from Table 1. In a more specific embodiment, the NIM has a combination of specific basal medium and additives shown in Table 1.

[0116] In Table 1, “CTS-DMEM / F12” refers to CTS TM KnockOut TM DMEM / F12 medium (Gibco); “DMEM / F12” refers to cGMP grade DMEM / F12 (Gibco); “CTS-NB” refers to CTS TM Neurobasal TM Culture medium (Gibco); "CTS-B27" refers to CTS TM Grade Vitamin A-Free Xeno-Free B-27 TM Additives (Gibco); "CTS-N2" refers to CTS TM N-2 Supplement (Gibco); and “CTS-GlutaMAX” refers to CTS TM GlutaMAX TM -I Supplement (Gibco); NDS refers to the combination of Noggin, Dorsomorphin and SB431542. The percentages in the table are calculated based on the volume of the culture medium.

[0117] Table 1. Preferred combinations of basal media and additives for EB culture medium and NIM

[0118]

[0119] In another embodiment, more than one culture medium of the present invention for the corresponding steps can be used during the culture of EB and RONA. In the EB culture process of step (2), two or more EB culture media of the present invention can be used in any order and any combination, such as an EB culture medium comprising the basal culture medium and additives listed in Table 1. Similarly, in the RONA formation process of step (3), two or more NIMs of the present invention can be used in any order and any combination, such as NIMs comprising the basal culture medium and additives listed in Table 1. For example, the first culture medium is used successively, and then the second culture medium is used; or the first culture medium is used first, then the second culture medium is used, and then the first culture medium is changed back; or the first culture medium is used first, then the second culture medium is used, and then the third culture medium is used; and so on. For example, in step (2) and / or step (3), a CTS or GMP grade N2M culture medium can be used first, and then a CTS grade NDM culture medium can be used. When more than one culture medium is used, the replacement of the culture medium can be carried out at any time point in a manner well known to those skilled in the art.

[0120] In the context of the present application, NPC medium is a medium for culturing NPCs. The NPC medium preferably comprises or consists of the following components: (i) a basal medium suitable for maintaining NPCs culture; and (ii) a CTS TM GlutaMAX TM -I additives and CTS without vitamin A TM The non-xenogeneic B-27 TM Additives. In a more preferred embodiment, the NPC culture medium further comprises BDNF, and / or GDNF, and / or L-ascorbic acid, and / or DB-cAMP.

[0121] Neurotrophic factors including BDNF and GDNF are important substances for the survival, growth or differentiation of dispersed neuronal populations. The BDNF or GDNF contained in NPC culture medium can use animal-free products. BDNF and / or GDNF can also be clinical grade, GMP grade, cGMP grade or CTS grade. TM class.

[0122] Any specific product exemplified in the present disclosure, such as culture medium, additive, inhibitor, neurotrophic factor or other reagent can be replaced by a product of the same type with a different trademark or product name, or other functional equivalents. Those skilled in the art will understand that substantially the same products under different trademarks or product names, as well as functional equivalents, are also included within the scope of the present invention.

[0123] Method steps

[0124] For the purpose of convenient reference only, the method of the present application can be divided into multiple steps, each of which includes one or more culture processes and / or treatments. Steps (1) to (5) of the method are basically divided based on the changes in the culture medium and / or the form of the cell culture obtained at the end of each step.

[0125] The step (1) of the method forms EBs by culturing stem cells in hPSC culture medium. In one embodiment, stem cells are cultured in a culture container such as a culture plate or a culture bottle, preferably an ultra-low adsorption culture plate, more preferably an ultra-low adsorption 96-well culture plate. Generally speaking, the culture of step (1) is carried out at 37 ° C. The cells are cultured for 24 to 72 hours, preferably 2 days, and then proceed to the next step. At the end of step (1), stem cells form EBs, and the formed EBs have morphological characteristics of a spherical shape and a smooth surface. The hPSC culture medium preferably contains a ROCK inhibitor.

[0126] The step (2) of the method cultured EBs in EB culture medium, and EBs undergo spontaneous neural differentiation. Specifically, in step (2), EBs are cultured in a suspension culture. In one embodiment, EBs are cultured in a culture container such as a culture plate or a culture bottle, preferably a low adsorption culture plate, and more preferably a low adsorption 6-well culture plate. Generally speaking, the culture in step (2) is carried out at 37°C. EBs are cultured for about 100 to 140 hours, preferably about 5 days, and then they are advanced to the next step. Preferably, the EB culture medium is replaced with fresh culture medium every day. At the end of step (2), EBs have morphological characteristics of a spherical shape and a smooth surface, and an enlarged volume. The preferred EB culture medium further contains an inhibitor, which helps to induce neural differentiation of EB.

[0127] Step (3) of the method allows EBs grown to the desired stage to grow in NIM to induce the formation of RONAs. In one embodiment, EBs are attached to a culture surface coated with a clinical-grade ECM material, such as a culture plate, a culture bottle, or a culture dish. Generally speaking, the culture in step (3) is carried out at 37°C. The neural induction in step (3) usually lasts for about 5-20 days. At the end of step (3), EBs differentiate to form RONAs, which are three-dimensional columnar cell aggregates formed by clusters of rosette-like cells stacked together.

[0128] In step (4) of the method, the rosette-like cells in the RONAs are separated and suspended in NPC culture medium to form neurospheres. In one embodiment, the neural aggregates are cultured in a culture container such as a culture plate or a culture bottle, preferably a low-adsorption culture plate, and more preferably a low-adsorption 6-well culture plate. For example, the amount of cells inoculated in a low-adsorption 6-well culture plate at the beginning of step (4) is the amount of RONAs from a 6-well culture plate. Generally speaking, the culture in step (4) is carried out at 37°C. The neural aggregates are cultured for about 12-24 hours and then proceed to the next step. At the end of step (4), spherical neurospheres are formed, which have morphological characteristics of a spherical shape and a smooth surface.

[0129] Step (5) of the method allows the neurospheres to form a monolayer of NPCs in the NPC culture medium. In one embodiment, step (5) includes a step (5a) of dispersing the neurospheres and a step (5b) of culturing. The dispersion can be performed by enzyme treatment, or by other methods such as mechanical means. Step (5a) performed by enzyme treatment is intended to dissociate the cells by treating the neurospheres with digestive enzymes for 5-10 minutes. The neurospheres treated with enzymes are digested into single cells so that they can be plated and cultured in a predetermined number. Step (5b) of culturing is performed on a culture surface such as a culture plate or a culture bottle coated with clinical grade EMC material. The cells are cultured at approximately 0.5-4.0x10 6 cells / cm 2 , preferably 0.8-3.5x10 6 cells / cm 2 , more preferably 1.0x10 6 cells / cm 2 Generally, the culture in step (5b) is carried out at 37°C. The next day, it can be observed that the NPCs reach a confluence of 80%-100%.

[0130] After step (5), NPCs can be further cultured and passaged once or more times. Preferably, cells are passaged once every three to five days, for example, once every four days. When passaged, NPCs can be treated with digestive enzymes, washed and counted, and then transferred to a culture surface such as a culture plate or culture bottle coated with a clinical grade ECM material for culture, preferably a 6-well culture plate. Preferably, the culture medium is replaced once a day or every other day, for example, by replacing half the volume of the culture medium with fresh culture medium.

[0131] Prior to step (1), the method of the present invention may include maintaining the culture of stem cells. To initiate step (1), the stem cells in the maintenance culture medium are treated with mechanical means or digestive enzymes, washed, and counted. Those skilled in the art are able to select a suitable culture medium for maintenance culture. When using stem cells that can be obtained through commercial channels, the maintenance culture can be carried out according to the conditions recommended by the supplier.

[0132] use

[0133] The methods of the present invention can be used to produce NPCs suitable for clinical and preclinical applications. For example, NPCs produced by the methods of the present invention can be used in drug development, disease model establishment, preclinical and clinical research, and can also be used in existing therapies and new therapies under development.

[0134] NPCs produced by the method of the present invention can also be used as starting cells to differentiate into one or more different types of neural cells, particularly differentiated neural cells suitable for preclinical and / or clinical applications. For example, the differentiated neural cells can be neurons or glial cells such as astrocytes or oligodendrocytes. Example

[0135] Example 1. Differentiation of hNPCs from hESCs

[0136] The process steps of the method of the present application are summarized in Figure 1 The detailed steps and materials are described below.

[0137] Preparation of embryoid bodies (EBs)

[0138] Human embryonic stem cell (hESC) cell line H1 was purchased from Shanghai Azesi Biotechnology Co., Ltd. (product catalog number AC-2001002H1) and maintained in culture medium containing hPSC XF medium (Biological Industries, BI) was added to a 6-well plate. 1 mL CTS was added to each well. TM TrypLE TM Select enzyme (Gibco), digested for 6 to 10 minutes in a 37°C incubator with 5% CO2. After the cells were detached and harvested, ESCs were plated at 1.65×10 4 cells / cm 2 The density was inoculated into a culture plate coated with clinical grade Laminin-521 (BioLamina, MX521 / CT521), with each well containing 2.5 mL hPSC XF Medium (BI).

[0139] When the confluence reached 80% to 90%, the culture medium was discarded and 2 mL of CTS was added to each well. TM Wash the cells with DPBS (without calcium chloride and magnesium chloride) (Gibco). Add 1 mL CTS to each well. TM TrypLE TM Select enzyme (Gibco), digest for 6 to 10 minutes in a 37°C incubator with 5% CO2. Observe under a microscope, and when some cells begin to fall off the culture plate, add 3 mL of Digestion was terminated with hPSC XF medium (BI). After centrifugation, cells were incubated with 10 μM Rock inhibitor ( Y-27632, Wako Resuspend in hPSC XF Medium (BI) and count viable cells.

[0140] The cells were seeded at a density of 30,000 cells per well in ultra-low attachment 96-well culture plates, each well of which contained 10 μM Rock inhibitor ( Y-27632, Wako) 200 μL hPSC XF medium (BI) was used, and the cells were cultured in a 37°C incubator containing 5% CO 2 . The next day, the formation of embryoid bodies (EBs) was observed in each well, and the EBs were of the same size.

[0141] Directed differentiation of EBs

[0142] Cells were cultured in the presence of 10 μM Rock inhibitor (Wako). After culturing in an ultra-low-adsorption 96-well plate containing hPSC XF medium (BI) for 2 days (day 0 to day 2 of directed differentiation), EBs were transferred to a low-adsorption 6-well plate at a density of 28 to 32 EBs per well, and the old medium was discarded. 2.5 mL of CTS-EB medium was added to each well, wherein the CTS-EB medium was composed of CTS TM KnockOut TM DMEM / F-12 medium (Gibco) and CTS TM Neurobasal TM The culture medium (Gibco) was mixed with basal medium at a ratio of 1:1 (vol / vol) and supplemented with 50 ng / mL Noggin GMP (R&D), 1 μM Dorsomorphin (Tocris), 10 μM SB431542 (Tocris), and 1 vol% CTS TM N-2 additive (100x) (Gibco) and 0.5 vol% CTS TM GlutaMAXTM -I supplement (200x) (Gibco) was prepared. EBs were cultured in a 37°C incubator containing 5% CO 2 for 5 days, and the medium was completely replaced with fresh CTS-EB medium every day.

[0143] On day 7 after differentiation, EBs were transferred to 6-well culture plates coated with clinical-grade Laminin-521 (BioLamina, MX521 / CT521) to allow EBs to fully adhere, and cultured in clinical-grade serum-free neural induction medium (NIM) to promote the expansion of neural stem cells and NPCs. Clinical-grade serum-free NIM was provided by CTS TM KnockOut TM DMEM / F-12 medium (Gibco) and CTS TM Neurobasal TM Culture medium (Gibco) was mixed with basal medium at a ratio of 1:1 (vol / vol) and supplemented with 1 vol% CTS TM N-2 additive (100x) (Gibco) and 0.5 vol% CTS TM GlutaMAX TM -I additive (200x) (Gibco) is prepared. In the first five days, half of the old culture medium is replaced with fresh NIM every other day, and half of the old culture medium is replaced with fresh NIM every day after five days. From the first day when EBs are inoculated into culture plates coated with clinical grade Laminin-521 (BioLamina, MX521 / CT521), EBs attach to the culture plates and gradually form adherent cell aggregates. Thereafter, a typical neural-specific rosette structure is formed, indicating the formation of neural stem cell aggregates. As neural stem cells and NPCs gradually expand, RONAs are formed in the center of the colony.

[0144] On day 21 after differentiation, neural aggregates rich in NPCs were selected and separated under a stereomicroscope, and the selected neural aggregates were transferred to a low-adhesion 6-well culture plate, each well of which contained 2.5 mL of CTS-NPC medium. The CTS-NPC medium was composed of CTS TM Neurobasal TMThe culture medium (Gibco) was supplemented with 20 ng / mL animal-free recombinant human / mouse / rat BDNF (Animal-Free Recombinant Human / Murine / Rat BDNF) (PeproTech), 20 ng / mL animal-free recombinant human GDNF (Animal-Free Recombinant Human GDNF) (PeproTech), 0.2 mM L-ascorbic acid (VC) (Sigma), 0.5 mM N 6 ,O 2 '-Dibutyryladenosine 3',5' cyclic monophosphate sodium salt (DB-cAMP) (Sigma), 1 vol% CTS TM GlutaMAX TM -I additive (100x) (Gibco) and 2 vol% CTS TM Xeno-Free B-27 Without Vitamin A TM Additive (50x) (Gibco) was prepared.

[0145] After 1 day of culture, the formation of neurospheres can be observed. Select the neurospheres from one well of the low-adhesion 6-well plate and transfer them to one well of a 24-well cell culture plate. Carefully discard the old culture medium. Add 0.5 mL CTS to each well. TM TrypLE TM Select enzyme (Gibco), digest the neurospheres for 5 to 10 minutes. When the outer layer of the neurospheres begins to become loose, gently add 1.5 mL of CTS-NPC medium to stop the digestion. Transfer the neurospheres to a 15 mL centrifuge tube and let them settle naturally for 2 to 5 minutes. Remove as much supernatant as possible. Add 1 mL of CTS-NPC medium, break the neurospheres into single cells with a 1 mL pipette tip, and count the live cells. Single cells are counted at 1.0x10 6 cells / cm 2 The cells were seeded at a density of 100 μg / mL onto a 6-well plate coated with clinical-grade Laminin-521 (BioLamina, MX521 / CT521), each well containing 5 mL of CTS-NPC medium. The next day, the isolated hNPCs were observed to grow in an adherent form with a confluence of 90% to 100%. Half of the old medium was replaced with fresh medium every day or every other day.

[0146] hNPCs grown in 6-well culture plates were digested and passaged every four days. Specifically, 2 mL CTS was first added to each well. TM hNPCs were washed once with DPBS (Dulbecco's phosphate buffered saline without calcium chloride and magnesium chloride) (Gibco), and then 1 mL of CTS was added to each well. TMTrypLE TM Select enzyme (Gibco), digest for 6 to 10 minutes in a 37°C incubator with 5% CO2. When cells begin to round up and remain attached to the culture plate under a microscope, discard CTS immediately. TM TrypLE TM Select enzyme solution. Add 3 mL of fresh CTS-NPC medium to each well and pipette 8-10 times with a 1 mL pipette tip until all cells are detached. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 200 g for 4 minutes. After centrifugation, discard the supernatant, resuspend the cell pellet in CTS-NPC medium, and count the live cells. Then, use 1.0x10 6 cells / cm 2 The cells were seeded at a density of 500 μg / mL onto a 6-well culture plate coated with clinical-grade Laminin-521 (BioLamina, MX521 / CT521), with each well containing 5 mL of CTS-NPC medium. The next day, hNPCs were observed to grow in an adherent form with a confluence of 90% to 100%. Half of the old medium was replaced with fresh medium every day or every other day. After hNPCs were passaged four times, hNPCs at P5 can be frozen or used directly.

[0147] Example 2. Characterization of hNPCs differentiated from hESCs

[0148] In order to confirm whether hNPCs were successfully differentiated from ESCs by the method described in Example 1, the obtained cells were identified based on morphological observation and detection of cell-specific biomarkers.

[0149] Cell morphology

[0150] Figure 2 This is a bright field image of hNPCs obtained by directed differentiation of hESCs according to the method in Example 1. Figure 2 It can be seen that the hNPCs obtained by the method provided by the present invention grow adherently and have a typical hNPC morphology, that is, a relatively homogeneous columnar cell body and exhibit a rosette-like radial cell arrangement.

[0151] Immunofluorescence staining

[0152] The hNPCs obtained by the method described in Example 1 were identified by immunofluorescence staining. Figure 3 As shown in the figure, most of the differentiated cells expressed FOXG1 and Nestin, indicating that a large number of high-purity hNPCs were successfully differentiated. + The cell ratio is as high as 98.52%, Nestin+ The cell ratio is as high as 99.26% ( Figure 3 ).

[0153] Flow cytometry

[0154] The cells differentiated in Example 1 were subjected to flow cytometry to identify hNPCs based on the expression of FOXG1, PAX6, and Nestin, and similar results to those obtained with the above-mentioned immunofluorescence staining were obtained. + The proportion of cells was as high as 95.69%, PAX6 + The cell ratio is as high as 76.11%, Nestin + The cell ratio is as high as 98.02% ( Figure 4 ).

[0155] Example 3. Differentiation of hESCs-derived hNPCs

[0156] Various experiments were performed to examine the ability of the hNPCs differentiated in Example 1 to further differentiate into desired cell types.

[0157] Differentiation of hNPCs into neurons

[0158] hNPCs grown in 6-well culture plates were digested and seeded for neuronal differentiation. CTS-neural differentiation medium was the same as the CTS-NPC medium described in Example 1. First, 2 mL of CTS was added to each well. TM hNPCs were washed once with DPBS (Dulbecco's phosphate buffered saline without calcium chloride and magnesium chloride) (Gibco), and then 1 mL CTS was added to each well. TM TrypLE TM Select enzyme (Gibco), digest for 6 to 10 minutes in a 37°C incubator with 5% CO2. When cells begin to round up and remain attached to the culture plate under a microscope, discard CTS immediately. TM TrypLE TM Select enzyme solution. Add 3 mL of fresh CTS-Neural Differentiation Medium to each well. Use a 1 mL pipette to pipette 8-10 times until all cells are detached. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 200 g for 4 minutes. After centrifugation, discard the supernatant, resuspend the cell pellet in CTS-Neural Differentiation Medium, and count the live cells. Then, use 2.5x10 4The cells were seeded into a 24-well culture plate coated with clinical-grade Laminin-521 (BioLamina, MX521 / CT521) at a density of 100 viable cells, with each well containing 500 μL CTS-neural differentiation medium. The next day, hNPCs were observed to grow in an adherent form. Half of the old culture medium was replaced with fresh culture medium every 3-7 days.

[0159] Immunofluorescence staining

[0160] The in vitro differentiation of hNPCs produced in Example 1 of the present invention can be identified by immunofluorescence staining.

[0161] Figure 5A The results of immunofluorescence staining of hNPCs after 2 months of in vitro differentiation were shown, detecting the expression of six-layer cortical markers specific to the human brain. Figure 5A As shown, the fluorescent signals represent the expression of various markers as follows: Brain-2 (BRN2) and special AT-rich sequence binding protein 2 (SATB2) for layers II to IV, chicken ovalbumin upstream promoter-transcription factor interacting protein 2 (CTIP2) for layers V and VI, T-box brain protein 1 (TBR1) for layers I, V and VI, and microtubule-associated protein 2 (Map2) for neurons. The results of immunostaining showed that the hNPCs obtained by the method of Example 1 have been successfully further differentiated into neural cells of all six layers of the human cerebral cortex.

[0162] Figure 5B The immunofluorescence staining results of synapsin and postsynaptic density protein 95 (PSD95) are shown, illustrating the expression of these two markers, which are presynaptic markers and postsynaptic markers, respectively. Figure 5B As shown in the figure, the signals representing synaptic proteins are distributed as discrete points, indicating that neurons have differentiated and matured. Figure 5C As shown in the figure, electrophysiological activity can be detected within one week of in vitro differentiation (day 6). In addition, after prolonged culture time (day 13), neuronal spike firing gradually increased, and more regular spontaneous firing, single electrode burst firing, and multi-electrode coordinated burst firing (network burst) were generated.

[0163] After the hNPCs produced in Example 1 of the present invention were differentiated in vitro for 3 months, immunofluorescence staining was performed to identify the expression of glial fibrillary acid protein (GFAP) and oligodendrocyte transcription factor 2 (OLIG2). Figure 5DAs shown, the positive signals of GFAP and OLIG2 indicate that the hNPCs obtained by the method of the present invention further differentiated into astrocytes and oligodendrocyte precursor cells.

[0164] In summary, the above immunofluorescence staining identification results prove that the hNPCs produced from ESCs by the method described in Example 1 can further differentiate into neural cells of all six layers of the human cerebral cortex and have corresponding physiological functions.

[0165] Growth curve

[0166] Figure 6 This is the growth curve of hNPCs (5th generation) produced in Example 1 of the present invention. According to the doubling time formula PDT = lg2 x (T-T0) / (lgN T -lgN0) to calculate the doubling time of hNPCs, where PDT is the doubling time, T is the time point for cell counting, T0 is the time point for cell inoculation, and N T is the value obtained by counting cells at time point T, and N0 is the number of cells inoculated. The doubling time of hNPCs was calculated to be 73 hours.

[0167] In addition, from Figure 6 It can be seen that hNPCs began to proliferate at 24 hours. At 72 hours, the number of cells was about twice the number of cells inoculated, and reached a plateau at 96 hours. Thereafter, the number of cells remained at a high level without increasing, indicating that the cells stopped proliferating. Cells at this stage still have the potential to differentiate in vitro. Therefore, subculturing cells after four days can enable the NPCs of this application to obtain the maximum yield.

[0168] Example 4. Differentiation of hNPCs from iPSCs

[0169] Production was performed in the same manner as described in Example 1, with the only difference being that human induced pluripotent stem cells (iPSCs) purchased from the Stem Cell Bank of the Chinese Academy of Sciences with catalog number SCSP-1301 were used as the starting cells for production instead of the hESC cell line H1.

[0170] Example 5. Characterization of hNPCs differentiated from iPSCs

[0171] In order to confirm whether hNPCs were successfully differentiated from iPSCs by the method shown in Example 4, the obtained cells were identified based on morphological observation and cell-specific biomarker detection.

[0172] Cell morphology

[0173] Figure 7 is a bright field image of hNPCs obtained by directed differentiation of iPSCs according to the method in Example 4 of the present invention. Figure 7 It can be seen that the hNPCs obtained by the method provided by the present invention grow adherently and have a typical hNPC morphology, that is, a relatively homogeneous columnar cell body and exhibit a rosette-like radial cell arrangement.

[0174] Immunofluorescence staining

[0175] The hNPCs obtained by the method described in Example 4 were identified by immunofluorescence staining. Figure 8 As shown in the figure, most cells expressed FOXG1 and Nestin, which are specific molecular markers of forebrain hNPCs, indicating that a large number of high-purity hNPCs were successfully differentiated. Specifically, in the differentiated cell population, FOXG1 + Nestin has a cell ratio of up to 95.70%. + The cell ratio is as high as 96.84% ( Figure 8 ).

[0176] Flow cytometry

[0177] The cells differentiated in Example 4 were subjected to flow cytometry to identify hNPCs based on the expression of FOXG1, PAX6, and Nestin, and similar results to those obtained with the above-mentioned immunofluorescence staining were obtained. + The proportion of cells was as high as 93.55%, PAX6 + The cell ratio is as high as 70.66%, Nestin + The cell ratio is as high as 95.16% ( Fig. 9 ).

[0178] Example 6. Differentiation of hNPCs derived from iPSCs

[0179] Various experiments were performed to examine the ability of the hNPCs differentiated in Example 4 to further differentiate into desired cell types such as neurons.

[0180] Differentiation of hNPCs into neurons

[0181] Differentiation of hNPCs was performed in the same manner as described in Example 3.

[0182] Immunofluorescence staining

[0183] The in vitro differentiation of hNPCs produced in Example 4 of the present invention can be identified by immunofluorescence staining.

[0184] Fig. 10AThe results of immunofluorescence staining of hNPCs after 2 months of in vitro differentiation were shown, detecting the expression of six-layer cortical markers specific to the human brain. Fig. 10A As shown, the fluorescent signals represent the expression of various markers as follows: BRN2 and SATB2 for layers II to IV, CTIP2 for layers V and VI, TBR1 for layers I, V and VI, and Map2 for neurons. The results of immunostaining showed that the hNPCs obtained by the method of Example 4 have been successfully differentiated into neural cells of all six layers of the human cerebral cortex.

[0185] Fig. 10B The immunostaining results of Synapsin and PSD95 are shown, illustrating the expression of these two markers, which are presynaptic and postsynaptic markers, respectively. Fig. 10B As shown in the figure, the signals representing synaptic proteins are distributed as discrete points, indicating that neurons have differentiated and matured. Fig. 10C As shown, electrophysiological activity can be detected on day 6 of in vitro differentiation. In addition, as the culture time increases, neuronal spike discharges gradually increase, and more regular spontaneous discharges, single electrode burst discharges, and multi-electrode coordinated burst discharges are generated.

[0186] After the hNPCs differentiated in Example 4 of the present invention were further differentiated in vitro for 3 months, immunofluorescence staining was performed to identify the expression of GFAP and OLIG2. Fig. 10D As shown, the positive signals of GFAP and OLIG2 indicated that at this time point, the hNPCs obtained by the method of the present invention further differentiated into astrocytes and oligodendrocyte precursor cells.

[0187] In summary, the above immunofluorescence staining identification results prove that the hNPCs produced from iPSCs by the method described in Example 4 can differentiate into neural cells of all six layers of the human cerebral cortex with desired physiological functions.

[0188] Example 7. Directed differentiation using different culture media

[0189] The inventors tested a series of different clinical-grade culture media in the method described in Example 1. Specifically, the composition of the basal culture media and additives of EB culture media and NIM was changed in each test while the other conditions remained unchanged. The results are summarized in Tables 2 and 3 below.

[0190] The medium combinations 1 to 4 in Tables 2 and 3 are the same as those listed in Table 1, and the combinations 5 to 10 are other medium compositions tested, wherein the EB medium and NIM in combinations 6 to 10 use different basal medium and additives. "NEAA" refers to GMP-grade MEM non-essential amino acid solution (MEM Non-Essential Amino Acids Solution); "CTS-KOSR" refers to KnockOut TM SR XenoFree CTS TM BI-EB means hPSC XF Medium (without growth factors).

[0191] Table 2. Test results of EB medium and NIM with different compositions

[0192]

[0193] Table 3. Detailed test results of different compositions of EB medium and NIM

[0194]

[0195]

[0196] Example 8. Initiation of directed differentiation using stem cell colonies

[0197] In a preferred embodiment of the present application, for example, in the method described in Example 1 or Example 4, EBs are formed by digesting stem cells and seeding the cells at a certain density in a culture plate. In this example, the inventors tested a method of seeding with stem cell colonies instead of a certain number of single cells. This example refers to the method in the article previously published by Xu et al. in 2016, and the culture medium and additives are replaced with corresponding clinical-grade reagents.

[0198] Maintain ESCs or iPSCs in CTS TM Vitronectin-coated culture plates were used hPSC XF medium. Treat ESCs or iPSCs colonies with collagenase NB 6GMP grade (0.15PZ U / mL HBSS, calcium, magnesium, no phenol red) for about 20 minutes. Without counting cells, the detached colonies are directly transferred to CTS-EBM and cultured in it for one day. CTS-EBM is composed of 20% KnockOut TM SR XenoFree CTS TM (Gibco), 1 vol% GMP grade MEM non-essential amino acid solution (Gibco), 0.5 vol% CTSTM GlutaMAX TM -I additive (Gibco) added to CTS TM KnockOut TM Prepared in DMEM / F-12.

[0199] The rest of the process is consistent with the steps in Example 1 and Example 4. From day 2 to day 6, the EB medium used to culture cells is CTS-EBM+NDS. CTS-EBM+NDS contains 50 ng / mL Noggin GMP, 1 μM Dorsomorphin, 10 μM SB431542, 20% KnockOut TM SR XenoFree CTS TM (Gibco), 1 vol% GMP grade MEM non-essential amino acid solution (Gibco), 0.5 vol% CTS TM GlutaMAX TM -I Additives (Gibco) and CTS TM KnockOut TM DMEM / F-12.

[0200] The EBs formed on day 6 were not spherical ( Fig.12 ), failed to differentiate into NPCs. The results show that the scheme that has been proven successful in the laboratory is uncertain in clinical translation. Simply replacing the basal culture medium and additives with corresponding clinical-grade reagents does not guarantee the successful generation of clinical-grade NPCs.

[0201] Example 9. Directed differentiation using two NIMs

[0202] The production was performed in the same manner as described in Example 1 or Example 4, with the only difference being that two NIMs were used to culture RONAs before and after to induce neural differentiation. On day 7 after the start of differentiation, EBs were transferred to a 6-well culture plate coated with clinical-grade Laminin-521 (BioLamina, MX521 / CT521) to allow the EBs to fully adhere, and cultured using two different clinical-grade serum-free neural induction media (NIM) to promote the expansion of neural stem cells and NPCs. The clinical-grade serum-free NIM used on day 1 to day 7 of RONAs differentiation was produced by CTS. TM KnockOut TM DMEM / F-12 medium (Gibco) and CTS TM Neurobasal TM Culture medium (Gibco) was mixed with basal medium at a ratio of 1:1 (vol / vol) and supplemented with 1 vol% CTS TMN-2 additive (100x) (Gibco) and 0.5 vol% CTS TM GlutaMAX TM Clinical-grade serum-free NDM used on days 8 to 14 of RONAs differentiation was prepared with CTS TM KnockOut TM DMEM / F-12 medium (Gibco) and CTS TM Neurobasal TM The culture medium (Gibco) was mixed with basal medium at a ratio of 1:1 (vol / vol) and supplemented with 0.5 vol% CTS TM N-2 additive (200x) (Gibco), 1 vol% CTS TM Xeno-Free B-27 Without Vitamin A TM Additive (100x) (Gibco) and 0.5 vol% CTS TM GlutaMAX TM -I additive (200x) (Gibco) was used to prepare the product.

[0203] The hNPCs obtained by the method described in this example were identified by immunofluorescence staining. Fig.13 As shown in the figure, most cells expressed FOXG1 and Nestin, indicating that a large number of high-purity hNPCs were successfully differentiated. Specifically, in the differentiated cell population, FOXG1 + Nestin has a cell ratio of up to 98.03%. + The cell ratio is as high as 99.30% ( Fig.13 ).

[0204] The in vitro differentiation of hNPCs produced in Example 9 of the present invention can be identified by immunofluorescence staining.

[0205] Fig.14A The results of immunofluorescence staining of hNPCs differentiated in vitro for 2 months were shown, detecting the expression of six-layer cortical markers specific to the human brain. Fig.14A As shown, the fluorescent signals represent the expression of various markers as follows: BRN2 and SATB2 for layers II to IV, CTIP2 for layers V and VI, and TBR1 for layers I, V, and VI. The results of immunostaining showed that the hNPCs obtained by the method of Example 9 have been successfully differentiated into neural cells of all six layers of the human cerebral cortex.

[0206] Fig. 14BThe immunostaining results of Synapsin and PSD95 are shown, illustrating the expression of these two markers, which are presynaptic and postsynaptic markers, respectively. Fig. 14B As shown in the figure, the signals representing synaptic proteins are distributed as discrete points, indicating that neurons have differentiated and matured. Fig. 14C As shown, electrophysiological activity can be detected on day 7 of in vitro differentiation. In addition, as the culture time increases, neuronal spike discharges gradually increase, and more regular spontaneous discharges, single electrode burst discharges, and multi-electrode coordinated burst discharges are generated.

[0207] After the hNPCs differentiated in the present invention were further differentiated in vitro for 3 months, immunofluorescence staining was performed to identify the expression of Map2, GFAP and OLIG2. Fig.14D As shown, the positive signals of Map2, GFAP and OLIG2 indicate that at this time point, the hNPCs obtained by the method of the present invention further differentiated into neurons, astrocytes and oligodendrocyte precursor cells.

[0208] In summary, the above immunofluorescence staining identification results prove that the hNPCs produced by the method described in Example 9 can differentiate into neural cells of all six layers of the human cerebral cortex with desired physiological functions.

Claims

1. A method for generating neural precursor cells (NPCs) from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), comprising: (1) Dispersing ESCs or iPSCs into single cells and culturing ESCs or iPSCs in human pluripotent stem cell medium (hPSC medium) to form embryoid bodies (EBs); (2) culturing the EBs formed by step (1) in an EB culture medium, wherein the EB culture medium comprises a basal culture medium, an additive, and an inhibitor, wherein the inhibitor is a BMP inhibitor, an AMPK inhibitor, and an ALK inhibitor, The basal medium and additives are selected from one of the following a) to d): a) the basal medium is a combination of KnockOut DMEM / F12 medium and Neurobasal medium, and the additives are N-2 additive, GlutaMAX-I additive and B-27 additive without vitamin A; b) the basal medium is a combination of KnockOut DMEM / F12 medium and Neurobasal medium, and the additives are N-2 additive and GlutaMAX-I additive; c) the basal medium is KnockOut DMEM / F12 medium alone, and the additives are N-2 additive, GlutaMAX-I additive, and B-27 additive without vitamin A; and d) the basal medium is a combination of DMEM / F12 medium and Neurobasal medium, and the additives are N-2 additive and GlutaMAX-I additive; (3) culturing the EBs after step (2) on a culture surface coated with an extracellular matrix (ECM) using a neural induction medium (NIM) to form rosette neural aggregates (RONAs), wherein the NIM comprises a basal medium and an additive, wherein the basal medium and the additive are selected from one of the following a)-d): a) the basal medium is a combination of KnockOut DMEM / F12 medium and Neurobasal medium, and the additives are N-2 additive, GlutaMAX-I additive and B-27 additive without vitamin A; b) the basal medium is a combination of KnockOut DMEM / F12 medium and Neurobasal medium, and the additives are N-2 additive and GlutaMAX-I additive; c) the basal medium is KnockOut DMEM / F12 medium alone, and the additives are N-2 additive, GlutaMAX-I additive, and B-27 additive without vitamin A; and d) the basal medium is a combination of DMEM / F12 medium and Neurobasal medium, and the additives are N-2 additive and GlutaMAX-I additive; (4) culturing the RONAs formed by step (3) in NPC medium or NIM medium to form neurospheres; and (5) The neurospheres were dispersed into single cells and cultured on a culture surface coated with ECM using NPC medium to form a monolayer of NPCs; wherein the embryonic stem cells are embryonic stem cells obtained from a commercial source, The basal culture medium of the NPC culture medium in step (4) and step (5) includes Neurobasal culture medium, and the additives are GlutaMAX-I additive and B-27 additive without vitamin A.

2. The method of claim 1, wherein the hPSC culture medium is supplemented with a ROCK inhibitor.

3. The method according to claim 1, wherein the inhibitor is Noggin, Dorsomorphin and SB431542.

4. The method of claim 1, wherein the NPC culture medium further comprises animal component-free brain-derived neurotrophic factor (BDNF), animal component-free glial cell line-derived neurotrophic factor (GDNF), L-ascorbic acid, and N 6 ,O 2’ -Dibutyryladenosine 3', 5' cyclic monophosphate sodium salt (DB-cAMP).

5. The method of claim 1, wherein in step (1), the dispersion of ESCs or iPSCs into single cells or cell aggregates is performed by digestive enzymes or by mechanical means.

6. The method of claim 1, wherein in step (2), more than one EB culture medium is used, and / or in step (3), more than one NIM is used.

7. A method for generating neurons, astrocyte precursor cells, astrocytes, oligodendrocyte precursor cells, oligodendrocytes or a mixed cell population comprising any one or more of these cells, comprising generating NPCs by the method described in any one of claims 1-6, and using the NPCs to generate the neurons, astrocyte precursor cells, astrocytes, oligodendrocyte precursor cells, oligodendrocytes or a mixed cell population comprising any one or more of these cells.

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