WO2024251119A1 - 一种免疫豁免性诱导性多能干细胞分化的神经细胞及其应用 - Google Patents

一种免疫豁免性诱导性多能干细胞分化的神经细胞及其应用 Download PDF

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WO2024251119A1
WO2024251119A1 PCT/CN2024/097338 CN2024097338W WO2024251119A1 WO 2024251119 A1 WO2024251119 A1 WO 2024251119A1 CN 2024097338 W CN2024097338 W CN 2024097338W WO 2024251119 A1 WO2024251119 A1 WO 2024251119A1
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cells
cell
culture medium
dopaminergic
fusion protein
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李翔
朱珉喆
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Xellsmart Biomedical Suzhou Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention belongs to the intersection field of genetic engineering and stem cell technology, and specifically relates to a neural cell differentiated from immune-immune induced pluripotent stem cells and an application thereof.
  • Stem cells are a type of "seed” cells that have the ability to self-renew and differentiate into specific functional somatic cells. Based on the degree of differences in stem cell characteristics, stem cells are mainly divided into: totipotent stem cells, pluripotent stem cells (PSCs) and adult stem cells. Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) have the potential to proliferate indefinitely, self-renew and differentiate into various types of cells, and have good application prospects in the treatment of cancer, neurological, cardiovascular and other related diseases.
  • PSCs pluripotent stem cells
  • hESC Human embryonic stem cells
  • iPSC induced pluripotent stem cells
  • Parkinson's disease is a serious neurodegenerative disease, and its overall incidence is second only to Alzheimer's disease (AD).
  • AD Alzheimer's disease
  • DAP transplanted dopaminergic neurons
  • the present invention successfully constructs B2M/CIITA biallelic knockout positive clones (DKO cells) by knocking out ⁇ -2-microglobulin (B2M) in the endoplasmic reticulum of induced pluripotent stem cells (iPSCs) and knocking out CIITA, a positive regulator of MHC-II gene transcription; then a lentiviral vector is used to overexpress a new gene based on the fusion of CD47 and CD24 domains in DKO cells, and the induced pluripotent stem cells thus obtained can further escape the killing of NK cells on the basis of escaping T cell attacks.
  • the cells are differentiated to obtain isolated immune-exempt dopaminergic nerve cells.
  • the dopaminergic nerve cells can significantly reduce or escape the recognition and attack of the immune system, especially the attack of natural killer cells, macrophages, etc., and effectively treat Parkinson's disease.
  • the present invention adopts the following technical solution:
  • the present invention provides an isolated low-immunogenic dopaminergic neural cell differentiated from an immunoprivileged induced pluripotent stem cell (i.e., a low-immunogenic iPS cell), comprising:
  • HLA-I and HLA-II human leukocyte antigens are reduced or absent;
  • the cells can escape T cell attack and NK cell killing.
  • gene editing tools are used in the immune privileged induced pluripotent stem cells to target one or more genes encoding one or more transcriptional regulators of HLA-I, and one or more genes encoding one or more transcriptional regulators of HLA-II, to achieve reduced or no expression of HLA-I and HLA-II genes.
  • the transcriptional regulatory factor of HLA-I may be preferably selected from: one or more of B2M, TAP1, TAP2, TAP-associated glycoprotein (Tapasin) or NLRC5; the transcriptional regulatory factor of HLA-II may be preferably selected from: one or more of CIITA, RFXANK, RFX5, RFXAP;
  • the transcriptional regulatory factors are preferably B2M and CIITA.
  • the immunoprivileged induced pluripotent stem cells further comprise a genetic modification targeting the CIITA gene by a rare-cutting endonuclease that selectively inactivates the CIITA gene.
  • the immunoprivileged induced pluripotent stem cells further include selectively inactivating B2M Genetic modification of the B2M gene by targeting a rare-cutting endonuclease.
  • the genetic modification for targeting the CIITA gene or the B2M gene by a rare-cutting endonuclease comprises a CAS protein or a polynucleotide encoding a CAS protein, and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene or the B2M gene.
  • the CRISPR/CAS9 system is used to directly knock out the B2M and CIITA exon segments at both ends, respectively, wherein the target sequences of the gRNA for the B2M gene are SEQ ID NO: 2 and SEQ ID NO: 3, and the target sequences of the gRNA for the CIITA gene are SEQ ID NO: 4 and SEQ ID NO: 5.
  • the expression of HLA-I and/or HLA-II genes is reduced or eliminated in the immunoprivileged induced pluripotent stem cells by introducing gene expression modifying molecules targeting one or more genes encoding one or more transcriptional regulators of HLA-I, or one or more genes encoding one or more transcriptional regulators of HLA-II, wherein the gene expression modifying molecules include one selected from siRNA, shRNA, microRNA, antisense RNA and another RNA-mediated inhibitory molecule.
  • the functional domain of CD47 in the fusion protein comprising the functional domains of CD47 and CD24 is the CD47 transmembrane domain; preferably, the amino acid sequence of the CD47 transmembrane domain is as shown in any one of SEQ ID NO: 6-10.
  • the functional domain of CD24 in the fusion protein comprising the functional domains of CD47 and CD24 is the signal peptide sequence of CD24, the mature peptide of CD24, the extracellular peptide of CD24, the membrane anchoring sequence of CD24, and the extracellular mature peptide of CD24; preferably, the amino acid sequence of the functional domain of CD24 is as shown in any one of SEQ ID NO: 11-16.
  • the fusion protein constructed by the functional domains of CD47 and CD24 is a protein that connects the CD47 SIRP ⁇ binding domain and the membrane anchoring sequence of CD24.
  • the fusion protein constructed by the functional domains of CD47 and CD24 is a protein in which the SIRP ⁇ binding domain of CD47 is inserted into the linking site of the extracellular peptide and membrane anchor sequence of CD24.
  • the fusion protein constructed by the functional domains of CD47 and CD24 is obtained by linking CD47 to the extracellular peptide of CD24.
  • the fusion protein constructed by the functional domains of CD47 and CD24 is obtained by linking the CD24 mature peptide to the SIRP ⁇ binding domain of CD47.
  • the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the extracellular mature peptide of CD24 is inserted into the SIRP ⁇ binding domain and the transmembrane domain connection point of CD47.
  • the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein of CD24.
  • the mature peptide sequence is connected to the CD47 transmembrane domain;
  • the amino acid sequence of the fusion protein constructed by the functional domains of CD47 and CD24 has more than 70% homology with the sequence shown in SEQ ID NO: 1, such as more than 80% homology, and more than 90%, more than 95%, or more than 98% homology;
  • amino acid sequence of the fusion protein constructed by the functional domains of CD47 and CD24 is as shown in SEQ ID NO: 1.
  • the increased expression of the fusion protein constructed by the functional domains of CD47 and CD24 is produced by introducing at least one copy of the fusion protein gene into the iPSC under the control of a promoter in the immune privileged induced pluripotent stem cells.
  • nucleic acid sequence encoding the fusion protein constructed by the functional domains of CD47 and CD24 can be obtained based on the amino acid sequence of the fusion protein.
  • the isolated low immunogenic dopaminergic neural cells are selected from dopaminergic neural precursor cells, dopaminergic neural progenitor cells, immature dopaminergic neural cells and mature dopaminergic neural cells; preferably dopaminergic neural precursor cells.
  • the present invention provides a method for isolating low-immunogenic dopaminergic neural cells described in the first aspect by in vitro differentiation from immunoprivileged induced pluripotent stem cells, wherein endogenous ⁇ -2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated in the immunoprivileged iPSC cells; and a fusion protein constructed by the functional domains of CD47 and CD24 is expressed; preferably, the amino acid sequence of the fusion protein constructed by the functional domains of CD47 and CD24 has more than 70% homology with the sequence shown in SEQ ID NO: 1, for example, more than 80% homology, and for example, more than 90%, more than 95%, or more than 98% homology; further preferably, the amino acid sequence of the fusion protein constructed by the functional domains of CD47 and CD24 is as shown in SEQ ID NO: 1;
  • the method comprises:
  • Immunoprivileged induced pluripotent stem cells were plated in the form of clumps on a matrix pre-coated with laminin at a number of about 100,000 per well, and the cells were cultured using E8 complete medium. The cells were digested and passaged every 4 to 5 days during the culture process.
  • DAP cells dopaminergic neural precursor cells
  • the first culture medium is a neural basal culture medium containing 1 ⁇ M-20 ⁇ M SB431542, 1 ⁇ M-3 ⁇ M DMH1, 0.2 ⁇ M-1 ⁇ M CHIR99021 and 1 ⁇ M-1.75 ⁇ M SAG;
  • the first culture medium is a neural basal medium containing 10 ⁇ M SB431542, 2 ⁇ M DMH1, 0.6 ⁇ M CHIR99021 and 1 ⁇ M SAG;
  • the second culture medium is a neural basal medium containing 5 ⁇ M-15 ⁇ M Y27632, 1 ⁇ M-20 ⁇ M SB431542, 1-3 ⁇ M DMH1, 0.2 ⁇ M-1 ⁇ M CHIR99021 and 1 ⁇ M-1.75 ⁇ M SAG;
  • the second culture medium is a neurobasal medium containing 10 ⁇ M Y27632, 10 ⁇ M SB431542, 2 ⁇ M DMH1, 0.6 ⁇ M CHIR99021 and 1 ⁇ M SAG;
  • the third culture medium is a neural basal medium containing 0.8 ⁇ M-1.2 ⁇ M DMH1, 0.1 ⁇ M-0.5 ⁇ M SAG, and 80ng/mL-120ng/mL fibroblast growth factor 8b (FGF8b);
  • the third culture medium is a neurobasal medium containing 1 ⁇ M DMH1, 0.2 ⁇ M SAG and 100 ng/mL fibroblast growth factor 8b (FGF8b);
  • the fourth culture medium is a neurobasal medium containing 0.1 ⁇ M-0.5 ⁇ M SAG and 80ng/mL-120ng/mL FGF8b;
  • the fourth culture medium is a neurobasal medium containing 0.2 ⁇ M SAG and 100 ng/mL FGF8b;
  • the fifth culture medium is a neurobasal medium containing 5 ⁇ M-15 ⁇ M Y27632, 0.1 ⁇ M-0.5 ⁇ M SAG, and 80ng/mL-120ng/mL FGF8b;
  • the fifth culture medium is a neurobasal medium containing 10 ⁇ M Y27632, 0.2 ⁇ M SAG and 100 ng/mL FGF8b;
  • the neural basal culture medium is selected from at least one of IMDM (Iscove's Modified Dulbecco's Medium) culture medium, Eagle's Basal Medium (BME) culture medium, GMEM culture medium, MEM culture medium, DMEM culture medium, Ham's F-12 culture medium, RPMI1640 culture medium and Neurobasal culture medium.
  • IMDM Iscove's Modified Dulbecco's Medium
  • BME Eagle's Basal Medium
  • GMEM culture medium GMEM culture medium
  • MEM culture medium MEM culture medium
  • DMEM culture medium Ham's F-12 culture medium
  • RPMI1640 culture medium RPMI1640 culture medium
  • Neurobasal culture medium Neurobasal culture medium.
  • the neural basal medium consists of the following components: 98% (v/v) DMEM/F12 (Gibco), 1% (v/v) non-essential amino acids (Non-essential amino acid, Gibco) and 1% (v/v) N2 supplement (N2 supplement, Gibco).
  • the present invention provides a composition comprising the isolated low immunogenic dopaminergic nerve cells according to the first aspect or the isolated low immunogenic dopaminergic nerve cells prepared according to the method of the second aspect.
  • the composition comprises the isolated low-immunogenic dopaminergic neural cells described in the first aspect and one or more therapeutic agents, wherein the therapeutic agents include peptides, cytokines, small molecule compounds, macromolecules, ADCs, antibodies, nanoparticles, biosimilars, mRNA, traditional Chinese medicine, proteins, vaccines, checkpoint inhibitors, mitogens, growth factors, small RNAs, double-stranded RNA (dsRNA), mononuclear blood cells, feeder cells, feeder cell components or their replacement factors, vectors comprising one or more polynucleic acids of interest, antibodies, etc.
  • the therapeutic agents include peptides, cytokines, small molecule compounds, macromolecules, ADCs, antibodies, nanoparticles, biosimilars, mRNA, traditional Chinese medicine, proteins, vaccines, checkpoint inhibitors, mitogens, growth factors, small RNAs, double-stranded RNA (dsRNA), mononuclear blood cells, feeder cells, feeder cell components or their replacement factors, vectors compris
  • the present invention provides a method for treating a patient suffering from a neurodegenerative disease or condition, the method comprising administering a therapeutically effective amount of the isolated low immunogenic dopaminergic neural cells described in the first aspect of the present invention or the isolated low immunogenic dopaminergic neural cells prepared according to the method of the second aspect or the composition described in the third aspect.
  • the method wherein the composition further comprises a therapeutically effective carrier.
  • the method wherein the isolated hypoimmune dopaminergic neural cells are on a biodegradable scaffold.
  • the method wherein the administering comprises implantation or injection.
  • the method wherein the neurodegenerative disease or condition is selected from Parkinson's disease, Huntington's disease disease and multiple sclerosis.
  • the present invention provides the use of the isolated low immunogenic dopaminergic nerve cells described in the first aspect, or the isolated low immunogenic dopaminergic nerve cells prepared according to the method of the second aspect, or the composition described in the third aspect in the preparation of a product for the treatment of neurodegenerative diseases or conditions.
  • the use, wherein the neurodegenerative disease or condition is selected from Parkinson's disease, Huntington's disease and multiple sclerosis; preferably Parkinson's disease.
  • the present invention inactivates the major histocompatibility complex MHC-I and class II genes in induced pluripotent stem cells, overexpresses the fusion protein XSG006 constructed by the functional domains of CD47 and CD24, and the obtained induced pluripotent stem cells are differentiated to obtain isolated low-immunogenic dopaminergic neural precursor cells, which can further escape the killing of NK cells and macrophages on the basis of escaping T cell attack.
  • the low-immunogenicity dopaminergic neural precursor cells separated by the present invention can effectively escape the attack of the immune system in vivo (humanized mice and non-human primates).
  • the low-immunogenicity dopaminergic neural precursor cells separated by the present invention were transplanted.
  • the test results after 4 months showed that the number of mouse rotations in the apomorphine (APO)-induced rotation experiment was significantly reduced, and it was statistically significant; in the cylinder experiment, the number of times the mouse's right forelimb hit the wall was significantly increased, basically restored to the level before modeling, and it was statistically significant; in the rotating rod experiment, the time the mouse stayed on the rotating rod was significantly increased, and it was statistically significant.
  • APO apomorphine
  • the monkey low-immunogenic dopaminergic neural precursor cells isolated by the present invention can survive and produce activity in the host body for a long time.
  • Allogeneic DAP cell transplantation was performed on non-human primates (NHP) with a complete immune system.
  • NEP non-human primates
  • monkey DKO+G6-DAP cells have low immunity and can escape the attack of the immune system, and survive for a long time in the brains of allogeneic monkeys with a complete immune system.
  • In vivo safety testing showed that low-immunogenic dopaminergic neural precursor cells (DKO+G6-DAP) have no difference in proliferation ability compared with unedited cells (WT-DAP), and both contain very small amounts of Ki67, indicating a low risk of tumorigenicity in vivo.
  • low-immunogenic DAP cells can differentiate into TH+, i.e., functional dopaminergic neurons in vivo.
  • FIG. 1 Schematic diagram of the CRISPR/CAS9 gene knockout strategy of B2M.
  • FIG. 1 Schematic diagram of CIITA’s CRISPR/CAS9 gene knockout strategy.
  • FIG. 1 Schematic diagram of the structure of pGC-EF1a plasmid.
  • FIG4 is a graph showing the expression of OCT-4 in various DAP cells detected by flow cytometry in Example 1;
  • FIG5 is a graph showing the expression of FOXA2, PAX6, OTX2, and LMX1A in each DAP cell detected by flow cytometry in Example 1;
  • FIG6 is a graph showing the expression of OTX2, EN1, LMX1A and FOXA2 in each DAP cell detected by immunofluorescence in Example 1;
  • FIG7 is a graph showing the expression of HLA-I/II in various cells detected by flow cytometry after stimulating each DAP cell with INF-gamma in Example 1;
  • FIG8 Sequential rt-PCR identification of the DKO+G6 cell line constructed in Example 1 and its derived DAP cells.
  • T cells are the positive control for detecting HLA class I/II molecules
  • FIG9 is a graph showing the results of using RTCA to detect the killing of WT, DKO and DKO+G6 differentiated DAP cells by PBNK cells in Example 2;
  • FIG10 is a photograph showing the killing of WT, DKO and DKO+G6 differentiated DAP cells by MAC in Example 2;
  • FIG11 is a graph showing the results of using RTCA to detect the killing of MAC cells against WT, DKO and DKO+G6 differentiated DAP cells in Example 2;
  • FIG. 12 is a diagram showing the results of validating the immune escape effect of DAP cells differentiated from DKO+G6 in mice with human immune system in Example 2;
  • FIG. 13 is the result of apomorphine (APO)-induced rotation experiment in the PD mouse model after transplantation of WT-DAP injection in Example 3;
  • Figure 16 shows the results of immunofluorescence staining of brain sections of PD mice transplanted with cells for 4 months in Example 3;
  • 16B is a 4x, 20x, and 60x magnification of the portion cut out in 16A.
  • FIG18 is a graph showing the expression of OTX2, EN1, LMX1A and FOXA2 in DAP cells derived from various monkey stem cells detected by immunofluorescence in Example 4;
  • FIG. 19 MRI results of monkey brain before and after WT-DAP (left brain) and DKO+G6-DAP (right brain) cell transplantation in Example 4;
  • Figure 20 Statistical graph of the left and right food intake rate of PD monkeys before and after WT-DAP and DKO+G6-DAP cell transplantation in Example 4;
  • Figure 21 Schematic diagram of WT-DAP and DKO+G6-DAP cell transplantation in Example 5 and MRI results of monkey brain before and after transplantation (A); MRI detection of cell size statistics (B).
  • FIG 22 Immunofluorescence staining of mouse brain in Example 5 (A), co-staining of hNA (red) & Ki67 (green) & DAPI (blue), where hNA stains transplanted cells and Ki67 marks proliferating cells; statistics of immunofluorescence staining (B).
  • FIG. 23 The results of brain immunofluorescence staining after PD-NOG transplantation into DKO+G6-DAP cells in Example 5.
  • FIG. 23 The results of brain immunofluorescence staining after PD-NOG transplantation into DKO+G6-DAP cells in Example 5.
  • the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.
  • the experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.
  • immune-privileged pluripotent stem cells A detailed description of immune-privileged pluripotent stem cells, methods for producing them, and methods for using them are found in 202210806871.7 filed on July 8, 2022 and PCT/CN2023/083761 filed on March 24, 2023, and the disclosures including the sequence listing, drawings, and examples are incorporated herein by reference in their entirety.
  • pluripotent cell refers to a cell that can self-renew and proliferate while maintaining an undifferentiated state and can be induced to differentiate into a specialized cell type under appropriate conditions.
  • the term “pluripotent cell” encompasses embryonic stem cells and other types of stem cells, including fetal, amniotic or somatic stem cells.
  • Exemplary human stem cell lines include the H9 human embryonic stem cell line.
  • Other exemplary stem cell lines include those available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute HUES collection (described in Cowan, CA et al., New England J. Med. 350: 13. (2004), which is incorporated by reference (incorporated herein in its entirety)
  • pluripotent stem cells have the potential to differentiate into any of the three germ layers: endoderm (e.g., gastric junction, gastrointestinal tract, lung, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.), or ectoderm (e.g., epidermal tissue and nervous system tissue).
  • endoderm e.g., gastric junction, gastrointestinal tract, lung, etc.
  • mesoderm e.g., muscle, bone, blood, urogenital tissue, etc.
  • ectoderm e.g., epidermal tissue and nervous system tissue.
  • the term “pluripotent stem cell” also encompasses "induced pluripotent stem cells” or "iPSCs,” which are a type of pluripotent stem cell derived from non-pluripotent cells. Examples of parental cells include somatic cells that have been reprogrammed to induce a pluripotent undifferentiated phenotype in various ways.
  • iPS or iPSC cells can be produced by inducing the expression of certain regulatory genes or by applying certain proteins exogenously. Methods for inducing iPS cells are known in the art and are further described below. (See, for example, Zhou et al., Stem Cells 27(11):2667-74 (2009); Huangfu et al., Nature Biotechnol. 26(7):795 (2008); Woltjen et al., Nature 458(7239):766-770 (2009); and Zhou et al., Cell Stem Cell 8:381-384 (2009); each of which is incorporated herein by reference in its entirety).
  • the term "subject” or “patient” refers to any animal, such as a domestic animal, a zoo animal, or a human.
  • a “subject” or “patient” can be a mammal, such as a dog, a cat, a bird, a livestock, or a human.
  • Specific examples of “subjects” and “patients” include, but are not limited to, individuals (particularly humans) suffering from diseases or conditions related to the liver, heart, lungs, kidneys, pancreas, brain, nervous tissue, blood, bones, bone marrow, etc.
  • immunoprivileged induced pluripotent stem cells refer to pluripotent cells that retain their pluripotent characteristics and cause a reduced immune rejection reaction when transferred into an allogeneic host. In a preferred embodiment, the cells do not cause an immune response. Therefore, “immune privilege” refers to an immune response that is significantly reduced or eliminated compared to the immune response of the parental (i.e., "wild type” or "wt") cells before the immune engineering outlined herein. In many cases, the cells are immunologically low immunogenic, but still retain pluripotency and differentiation potential.
  • HLA human leukocyte antigen
  • MHC major histocompatibility complex
  • HLA-I major histocompatibility complex
  • HLA-I includes three proteins, HLA-A, HLA-B and HLAC, which present peptides from the inside of the cell, while the antigens presented by the HLA-I complex attract killer T cells (also known as CD8+T- cells or cytotoxic T cells).
  • B2M beta-2 microglobulin
  • HLA-II includes five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and HLA-DR, which present antigens to T lymphocytes from the outside of the cell. This stimulates CD4+ cells (also known as T helper cells).
  • MHC or “HLA” is not meant to be limiting, as it depends on whether the gene is from human (HLA) or mouse (MHC). Therefore, as it relates to mammalian cells, these terms can be used interchangeably herein.
  • CD47 is also called integrin-associated protein (IAP), which is widely expressed on the surface of various cells. It can interact with signal regulatory protein ⁇ (SIRP ⁇ ) and thrombospondin-1 (TSP-1), and plays an important role in mediating the oxidative stress response and immune repair process of certain cardiovascular diseases.
  • SIRP ⁇ signal regulatory protein ⁇
  • TSP-1 thrombospondin-1
  • CD47 contains three components: an amino-terminal extracellular immunoglobulin variable domain (IgV)-like domain, a 5-domain transmembrane region, and a carboxyl-terminal splice variant cytoplasmic tail.
  • IgV extracellular immunoglobulin variable domain
  • CD24 stands for cluster of differentiation 24, also known as heat-stable antigen. It is a highly glycosylated glycosylphosphatidylinositol-anchored surface protein, which is known to interact with Siglec-10 (sialic-acid-binding Ig-like lectin10) on innate immune cells to inhibit destructive inflammatory responses in response to infection, sepsis, liver injury and chronic graft-versus-host disease. CD24 is a small molecular weight sialic acid glycoprotein composed of 31 amino acids with a typical mucin structure and is located in lipid rafts through its glycosylated phosphatidylinositol (GPI) anchor.
  • GPI glycosylated phosphatidylinositol
  • Gene knockout herein refers to the process of inactivating a specific gene in the host cell in which it resides, resulting in the non-production of the protein of interest or the formation of an inactivated form. As will be appreciated by those skilled in the art and described further below, this can be accomplished in many different ways, including removing the nucleic acid sequence from the gene, or interrupting the sequence with other sequences, changing the reading frame, or changing the regulatory components of the nucleic acid. For example, all or part of the coding region of the gene of interest can be removed or replaced by a "nonsense" sequence, all or part of a regulatory sequence such as a promoter can be removed or replaced, a translation initiation sequence can be removed or replaced, etc.
  • Gene knock-in herein refers to the process of increasing genetic function to a host cell. This results in an increase in the level of the encoded protein. As will be appreciated by those skilled in the art, this can be achieved in several ways, including adding one or more other copies of the gene to the host cell or changing the regulatory components of the endogenous gene, thereby increasing the expression of the protein. This can be achieved by modifying the promoter, adding different promoters, adding enhancers or modifying other gene expression sequences.
  • Allogeneic in this context refers to the genetic dissimilarity between the host organism and the cell transplant, in which an immune response is generated.
  • identity percentage refers to two or more sequences or subsequences that have a specified percentage of identical nucleotides or amino acid residues when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or by visual inspection.
  • sequence comparison algorithms e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art
  • the "identity" percentage can exist over regions of the sequences being compared, such as over functional domains, or alternatively over the full length of the two sequences to be compared.
  • one sequence is typically used as a reference sequence to which a test sequence is compared.
  • test sequence and the reference sequence are entered into a computer, and subsequences are specified if necessary.
  • sequence coordinates are provided and sequence algorithm program parameters are specified.
  • sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.
  • Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., see below).
  • the BLAST algorithm is one example of an algorithm suitable for determining percent sequence identity and sequence similarity and is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).
  • homologous or derived sequences are biologically active molecules that are similar to reference molecules at the nucleotide sequence, peptide sequence, function or structural level. Homologues can include sequence derivatives that have a certain percentage of identity with the reference sequence. Therefore, in one embodiment, homologous or derived sequences have at least 70% sequence identity. In a specific embodiment, homologous or derived sequences have at least 80% or 85% sequence identity. In a specific embodiment, homologous or derived sequences have at least 90% sequence identity. In a specific embodiment, homologous or derived sequences have at least 95% sequence identity.
  • homologous or derived sequences have at least 50, 55, 60, 65, 70, 75, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity.
  • Homologous or derived nucleic acid sequences can also be defined by their ability to remain bound to a reference nucleic acid sequence under high stringency hybridization conditions.
  • a homologue having structural or functional similarity to a reference molecule may be a chemical derivative of the reference molecule. Methods for detecting, generating and screening structural and functional homologues and derivatives are known in the art.
  • Dopaminergic neural precursor cells herein generally refer to cells that can proliferate and/or differentiate into dopaminergic neurons in vitro or in vivo.
  • Dopaminergic neural precursor cells can be derived from ventral midbrain neurons and can be differentiated from pluripotent stem cells.
  • Dopaminergic neural precursor cells can also be differentiated or reprogrammed from other cell types.
  • the "dopaminergic nerve cell” herein is selected from dopaminergic nerve precursor cells, dopaminergic nerve progenitor cells, immature dopaminergic nerve cells and mature dopaminergic nerve cells; preferably, it is a dopaminergic nerve precursor cell.
  • Example 1 Differentiation of midbrain dopaminergic neural precursor (DAP) cells using low immunogenic stem cells (i.e., low immunogenic iPS cells)
  • WT, DKO and DKO+G6 cells were plated in culture flasks pre-coated with Matrigel. After 24 hours of culture, the cells were switched to pre-differentiation medium to induce differentiation into midbrain cells. After 9 days of differentiation, high-purity midbrain cells were obtained. Subsequently, the obtained midbrain cells were amplified by adding neural progenitor cell culture medium to obtain a large number of high-purity neural progenitor cells. Finally, neural progenitor cells were further differentiated into neural cells by adding neural precursor cell culture medium. The cells can differentiate into dopaminergic neural precursor (DAP) cells in vitro.
  • DAP dopaminergic neural precursor
  • the present invention uses human induced pluripotent stem cells (iPSC cells, human blood PBMCs are reprogrammed into iPSC cells, i.e., WT cells, through CTS TM CytoTune TM -iPS2.1 Sendai virus reprogramming kit (Cat.
  • iPSC cells human blood PBMCs are reprogrammed into iPSC cells, i.e., WT cells, through CTS TM CytoTune TM -iPS2.1 Sendai virus reprogramming kit (Cat.
  • B2M-gRNA1 and B2M-gRNA2 are used to directly knock out the B2M exon segment at both ends, and then two pairs of PCR primers B2M-F1/R1 and B2M-F2/R2 are used to verify the genome sequence knockout.
  • B2M-gRNA1 CGTGAGTAAACCTGAATCTT
  • B2M-gRNA2 AGTCACATGGTTCACACGGC
  • CRISPR/CAS9 gene knockout strategy of CIITA is shown in Figure 2 , where CIITA-gRNA1 and CIITA-gRNA2 were used to directly knock out both ends of the CIITA exon segment, and then two pairs of PCR primers, CIITA-F1/R1 and CIITA-F2/R2, were used to verify the genome sequence knockout.
  • CIITA-R1 CCTTCCATGTCACACAACAGCC
  • Neon transfection system 100 According to the Neon transfection system 100 ⁇ L electroporation system, add 15 ⁇ g TrueCut TM Cas9 Protein + 3 ⁇ g gRNA (B2MgRNA1 + B2MgRNA2 + CIITA gRNA1 + CIITA gRNA2) to form the RNP system, mix well and place at room temperature for 20 minutes.
  • qPCR was performed using the following B2M-F/R and CIITA-F/R primers to detect the expression of B2M and CIITA at the RNA level in the B2M/CIITA biallelic knockout clone DKO to confirm the knockout.
  • the fusion protein XSG006 was overexpressed in the DKO cells prepared in 1.2 by lentiviral infection to form the fusion protein stable cell line DKO+G6.
  • the fusion protein XSG006 is constructed by the functional domains of CD47 and CD24, which connects the mature peptide sequence of CD24 and the transmembrane domain of CD47, and its amino acid sequence is shown in SEQ ID NO.1.
  • nucleic acid sequence encoding XSG006 (the amino acid sequence of XSG006 is shown in SEQ ID NO: 1) was directly synthesized and constructed into a lentiviral plasmid (pGC-EF1a) driven by EF1a and carrying a puromycin selection marker, and the structure of the pGC-EF1a plasmid is shown in Figure 3.
  • the plasmid was digested with BamHI/NheI, and after successful ligation, Sanger sequencing was used to verify the correctness of the inserted sequence and perform virus packaging.
  • the DKO human induced pluripotent stem cells obtained in 1.2 were transfected and then screened with a medium containing puromycin.
  • XSG006 is a completely exogenous sequence.
  • the constructed DKO+G6 cells were tested for the insertion of the sequence in the genome using PCR. DKO cells were used as negative controls. After confirming that the expression was correct, cell amplification and subsequent Functional testing.
  • a kit for differentiating human induced pluripotent stem cells (WT cells), DKO cells and DKO+G6 cells into dopaminergic neural precursor cells comprising: a first culture medium, a second culture medium, a third culture medium, a fourth culture medium and a fifth culture medium;
  • the first culture medium is a neural basal medium containing 10 ⁇ M SB431542, 2 ⁇ M DMH1, 0.6 ⁇ M CHIR99021 and 1 ⁇ M SAG;
  • the second culture medium was a neurobasal medium containing 10 ⁇ M Y27632, 10 ⁇ M SB431542, 2 ⁇ M DMH1, 0.6 ⁇ M CHIR99021, and 1 ⁇ M SAG;
  • the third culture medium was a neurobasal medium containing 1 ⁇ M DMH1, 0.2 ⁇ M SAG, and 100 ng/mL fibroblast growth factor 8b (FGF8b);
  • the fourth culture medium is a neurobasal medium containing 0.2 ⁇ M SAG and 100 ng/mL FGF8b;
  • the fifth culture medium is a neurobasal medium containing 10 ⁇ M Y27632, 0.2 ⁇ M SAG, and 100 ng/mL FGF8b;
  • the neural basal medium is composed of the following components: 98% (v/v) DMEM/F12 (Gibco), 1% (v/v) non-essential amino acid (Non-essential amino acid, Gibco) and 1% (v/v) N2 supplement (N2 supplement, Gibco).
  • a method for differentiating human induced pluripotent stem cells (WT cells), DKO cells and DKO+G6 cells into dopaminergic neural precursor cells comprising the steps of using the kit of 2.1, specifically as follows:
  • Human induced pluripotent stem cells, DKO cells or DKO+G6 cells were plated in the form of clumps on a matrix pre-coated with Laminin at a number of about 100,000 per well, and the cells were cultured using E8 complete medium. The cells were digested and passaged every 4 to 5 days during the culture process;
  • the culture medium is replaced with the first culture medium in the kit of 2.1 (as the 0th day of differentiation, D0), and cultured in a carbon dioxide cell culture incubator at 37°C and 5% CO2 for 8 days (the medium is changed every two days);
  • DAP cells dopaminergic neural precursor cells
  • DAP dopaminergic neural precursor
  • the identity of the derived cells was confirmed by flow cytometry and immunofluorescence.
  • the expression of the stemness gene OCT4 in WT, DKO, and DKO+G6 differentiated DAP cells was verified to be ⁇ 1% ( Figure 4), confirming that they had exited pluripotency and were completely differentiated cells.
  • FACS was used to detect the expression of dopaminergic neural precursor cell marker genes FOXA2, PAX6, OTX2, and LMX1A in WT, DKO, and DKO+G6 differentiated DAP cells ( Figure 5).
  • Immunofluorescence was also used to detect the expression of dopaminergic neural precursor cell marker genes in WT, DKO and DKO+G6 derived cells.
  • the specific operation was to plate the cells in a 12-well plate, aspirate the culture medium after the cells grew to a density of 60-80%, and add 4% paraformaldehyde for fixation. After the cell membrane was broken, the primary antibodies of OTX2, EN1, LMX1A and FOXA2 were used for incubation at 4°C overnight. After washing away the primary antibodies, the secondary antibodies with fluorescent labels were incubated at room temperature, and then the fluorescence microscope was used for observation. The results are shown in Figure 6, and all cells expressed dopaminergic neural precursor cell marker genes.
  • INF-gamma was used to stimulate WT and DKO+G6 differentiated DAP cells: cells were plated in well plates, and the medium containing INF-gamma was added to the cells when the medium was changed the next day. After 48 hours, the cells were digested and the expression of HLA-I/II was detected by flow cytometry. The results are shown in Figure 7. After differentiation into DAP, the positive clones of DKO+G6 stem cells with B2M/CIITA biallelic knockout still did not express HLA-I/II, and could not upregulate HLA-I/II in response to INF-gamma stimulation.
  • the molecular sequence of XSG006 is a completely exogenous sequence.
  • RT-PCR was used to compare the stability of XSG006 expression between WT and dopamine neural progenitor cells differentiated from it, and between DKO+G6 and dopamine neural progenitor cells differentiated from it (Figure 8).
  • WT-DAP and DKO-DAP cells were used as negative controls. After confirming that the expression was correct, cell expansion and subsequent functional testing were performed.
  • Low immunogenic stem cells can be successfully differentiated into dopaminergic neural precursor (DAP) cells in vitro.
  • DAP dopaminergic neural precursor
  • the derived cells have the characteristics of DAP cells and retain the expression of low immunogenic molecules.
  • the human induced pluripotent stem cells selected by the present invention use CRISPR/CAS9 to knock out beta-2-microglobulin (B2M) in the endoplasmic reticulum, so that HLA-I on the cell surface cannot form functional molecules, thereby escaping the killing of allogeneic CD8+T cells; the killing of CD4+T cells is escaped by knocking out the positive regulator CIITA of HLA-II gene transcription, and reducing the expression of HLA-II class molecules.
  • XSG006 is expressed in cells (DKO) with B2M/CIITA biallelic knockout by lentivirus to escape the killing of innate immunity.
  • the cells successfully constructed are DKO+G6, which are low immunogenic stem cells.
  • the dopaminergic neural precursor cells (DKO+G6-DAP) derived from the low immunogenic stem cells of Example 1 should also have the ability to not activate the immune system and escape its killing.
  • NK activation factors were added to PBMC in advance to increase the NK ratio in PBMC and the killing performance of T cells.
  • Activated mixed lymphocytes (PBNK) were used as effector cells for RTCA experiments to comprehensively evaluate the immune escape ability of DKO+G6-DAP cells (PMID: 33309274). The specific operation was to perform PBNK cell killing assay on the XCelligence platform (ACEA BioSciences, San Diego, CA.).
  • WT-DAP, DKO-DAP and DKO+G6-DAP cells were resuspended in 100 ⁇ l cell-specific culture medium (Neurobasal TM culture medium, catalog number: 21103049, Thermofisher) and plated on a 96-well E-plate (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index value reached 1, T cells were added at an E:T ratio of 1:1. The data were standardized and analyzed using RTCA software (ACEA). The results are shown in Figure 9 , and only DKO+G6-DAP cells can escape the killing of PBNK.
  • WT, DKO and DKO+G6 differentiated DAP cells were plated in 12-well plates, and the culture medium was discarded after 24 hours, and MAC cells were added for co-culture. After 24 hours, the remaining WT, DKO and DKO+G6 differentiated DAP cells were observed after the removal of MAC cells, and the results are shown in FIG10 .
  • MAC cell killing assay was performed on the XCelligence platform (ACEA BioSciences, San Diego, CA.). WT-DAP, DKO-DAP, and DKO+G6-DAP cells were resuspended in 100 ⁇ l of cell-specific culture medium and plated on 96-well E-plates (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index value reached 1, MAC cells were added at an E:T ratio of 1:1. The data were normalized and analyzed using RTCA software (ACEA). The results are shown in Figure 11.
  • the survival of DAP cells in vivo was observed by observing the bioluminescence detection (BLI) injected into the brain of humanized mice.
  • the specific operation is: the lentivirus carrying luciferase (luc) is used to infect WT and DKO+G6 differentiated DAP cells, and then the cells expressing luc are transplanted into the brain of CD34+HSC-reconstructed humanized immune system mice.
  • luc's luminescent substrate D-luciferin A025011, Shanghai Yishen Biotechnology Co., Ltd.
  • an in vivo imager iVIS spectrum, PerkinElmer
  • the fluorescence of the transplanted site was continuously detected for 51 days.
  • the fluorescence value of the WT differentiated DAP cells tended to the background value, while the DKO+G6 differentiated After DAP cell transplantation, continuously rising fluorescence values can be detected, indicating that DKO+G6 differentiated DAP cells can effectively escape the attack of the immune system in vivo compared with WT differentiated DAP cells ( FIG. 12 ).
  • This embodiment describes a study on the use of in vitro differentiated dopaminergic neural precursor cells (DAP) from stem cells for the treatment of Parkinson's disease in a mouse model.
  • DAP dopaminergic neural precursor cells
  • Scid beige mice were used for modeling, and three behavioral tests, namely apomorphine-induced rotation, cylinder test, and rotarod test, were performed to select modeling mice that met the standards for cell transplantation.
  • the experiment set up a solvent (cell preservation solution, artificial cerebrospinal fluid ACSF, Maokang Bio, MX0951) control group and a test group (WT-DAP injection and DKO+G6-DAP injection).
  • mice After administration, the behavior of the three groups of mice was tested by immunofluorescence staining of brain tissue pathological sections and three behavioral tests, and the behavioral differences of the three groups of mice, the behavioral differences of the mice themselves before and after administration, and the changes in cell retention and dopaminergic neurons at the administration site were compared.
  • Scid beige mice were anesthetized with isoflurane gas anesthesia machine, and the anesthetized mice were fixed on the mouse brain stereotaxic instrument, and 6-hydroxydopamine (6-OHDA) solution (H4381-100mg (sigma)) was slowly injected into the substantia nigra of the left brain of the mice in stereotaxic manner.
  • 6-hydroxydopamine (6-OHDA) solution H4381-100mg (sigma) was slowly injected into the substantia nigra of the left brain of the mice in stereotaxic manner.
  • the cylinder test, the rotarod test and the apomorphine-induced rotation behavioral test were performed to determine the model mice.
  • the model mice were divided into groups for injection, and 2 ⁇ L WT-DAP injection solution, 2 ⁇ L DKO+G6-DAP injection solution and 2 ⁇ L cell preservation solution were slowly injected into the left striatum of the mouse brain in a stereotaxic manner. Behavioral tests were performed once a month after surgery for 4 months. After the last test, the brain of the mouse was sampled.
  • mice in the test group showed significant behavioral improvement at 4 months (p ⁇ 0.05), and the proportion of the number of times the right forelimb hit the wall increased significantly, indicating that transplantation of WT-DAP injection solution is effective in improving the Parkinson's-related resting tremor behavior of model mice ( Figure 14).
  • mice in the test group had significant behavioral improvements at 4 months (p ⁇ 0.05), and the time they stayed on the rotarod was significantly increased; compared with the control group, the behavioral improvements were also significant (p ⁇ 0.05). This shows that transplantation of WT-DAP injection is effective in improving the posture and gait disorder behavior of model mice (Figure 15).
  • Immunofluorescence and immunohistochemical staining of frozen sections of PD mouse brain tissue tissue sections were rinsed with PBS for 5 minutes, 3 times; blocked for 1 hour at 37°C; the blocking solution was a PBS solution containing 5% BSA and 0.3% triton-100.
  • Add primary antibody react slowly in a humidified box at 4°C for 24 hours to 48 hours; the primary antibody diluent was a PBS solution containing 1% BSA and 0.3% triton-100.
  • the fluorescent secondary antibody was incubated at room temperature in the dark for 1 to 2 hours. Then rinsed with PBS for 5 minutes, 3 times, and sealed with DAPI, and photographed under a microscope.
  • a vehicle control group cell preservation solution
  • a test group WT-DAP injection and DKO+G6-DAP injection
  • This example describes an in vitro study of iPSC-derived primate (NHP) models.
  • DAP Differentiated dopaminergic neural precursor cells
  • NAP Non-human primates
  • the methods include MRI scanning and analysis, PET analysis, video analysis, and behavioral analysis (see, for example, Kikuchi et al., Nature, 2017, 548, 592-596).
  • NHP non-human primate
  • DAP cell transplantation in non-human primates (NHPs) with Parkinson’s disease (PD) DAP differentiated cells generated from stem cells were evaluated in a monkey model of Parkinson’s disease (PD).
  • monkey stem cells were differentiated according to the method described above to generate monkey WT (monkey iPSC cells, monkey blood PBMCs were prepared by CTS TM CytoTune TM -iPS2.1 Sendai virus reprogramming kit (Cat. No.: A34546)) and monkey DKO+G6 differentiated DAP cells (monkey DKO+G6-DAP cells).
  • the B2M exon segment was directly knocked out at both ends using NHP-B2M-gRNA1 and NHP-B2M-gRNA2, and then the genomic sequence knockout was verified using NHP-B2M-F/R pair PCR primers, respectively.
  • the CIITA exon segment was directly knocked out at both ends using NHP-CIITA-gRNA1 and NHP-CIITA-gRNA2, and then the genomic sequence knockout was verified using NHP-CIITA-F/R pair PCR primers, respectively, and the monkey DKO+G6 cells overexpressed NHP-XSG006 protein.
  • Monkey DAP cells were transplanted stereotaxically into the putamen of MPTP-treated monkeys on both sides, with monkey WT-DAP cells injected on the left side and monkey DKO+G6-DAP cells injected on the right side.
  • MRI was used to determine the survival of DAP cells in the left and right brains ( Figure 19). MRI results showed that monkey DKO+G6-DAP cells persisted in the brain during the 28-day observation period, while monkey WT-DAP cells were no longer observed on the fifth day.
  • the effects of the transplanted DA neurons were evaluated according to methods recognized by those skilled in the art.
  • the changes or differences in left and right absorptive behavior, movement, PD symptoms, dopaminergic function, etc. were evaluated in monkeys using DAP cells differentiated from monkey WT and monkey DKO+G6 ( Figure 20).
  • the results of feeding rate showed that after the successful modeling, the two forelimbs did not have any feeding behavior before the cell transplantation.
  • the left forelimb (the side affected by the injection of monkey DKO+G6-DAP) has shown signs of improvement; the right forelimb (the side affected by the injection of WT-DAP) has no obvious improvement.
  • Example 5 Low immunogenic dopaminergic neural precursor cells in a normal non-human primate (NHP) model
  • This embodiment describes a study on the immune immunity of dopaminergic neural precursor cells (DAP) differentiated in vitro from monkey stem cells (iPSC) in a non-human primate (NHP) model with a complete immune system.
  • DAP dopaminergic neural precursor cells
  • iPSC monkey stem cells
  • NHS non-human primate
  • Allogeneic transplantation of NHP-DAP cells was performed on normal non-human primates without modeling to confirm that DAP derived from low immunogenic iPS cells can resist immune system attacks, and MRI scanning and analysis were used to monitor the survival of cells in vivo (see, for example, Kikuchi et al., Nature, 2017, 548, 592-596).
  • the same batch of monkey WT-DAP cells and monkey DKO+G6-DAP cells were transplanted in immunodeficient NOG mice to confirm the in vivo safety of DAP cells.
  • monkey DKO+G6-DAP cells were injected into PD-NOG mice to confirm the differentiation characteristics of low immunogenic
  • Monkey DAP cells were transplanted into the putamen in a stereotaxic manner, with monkey WT-DAP cells injected on the left side and monkey DKO+G6-DAP cells injected on the right side.
  • MRI was used to determine the survival of DAP cells in the left and right brains ( Figure 21A&B). MRI scans were performed on the monkey brains before transplantation (pre-TX), on the day of transplantation (day0), and on days 7, 14, 21, 28, 42, 56, 90, 122, 152, 183, and 212 after transplantation. The results showed that during the 259-day observation period, monkey DKO+G6-DAP cells continued to exist in the brain, while monkey WT-DAP cells could not be observed on day 14-21.
  • monkey DKO+G6-DAP cells have low immunity and can escape immune system attacks, and survive for a long time in the brains of allogeneic monkeys with a complete immune system; at the same time, unmodified WT-DAP cells are rejected and cannot survive in the same environment.
  • the monkey WT-DAP cells and monkey DKO+G6-DAP cells were stereoscopically injected into the left striatum of different NOG mice using the method of Example 3B.
  • the mice were sampled for brain sampling 2 months after transplantation, and the frozen sections of NOG mouse brain tissue were immunofluorescently stained with reference to the method of Example 3D.
  • the coronal frozen sections of the brain tissue of mice 3 months after cell transplantation were co-stained with hNA (red) & Ki67 (green) & DAPI (blue) ( Figure 22A).
  • Example 3A The method of Example 3A was used to model PD in NOG mice, and the model mice were stereotactically slowly injected with monkey DKO+G6-DAP cells into the left striatum.
  • Brain samples were taken from mice 3 months after transplantation, and frozen sections of brain tissue of PD mice were immunofluorescently stained with reference to the method of Example 3D. Coronal frozen sections of brain tissue of mice 3 months after cell transplantation were co-stained with hNA (red) & TH (green) & DAPI (blue) ( Figure 23). The staining results showed that low immunogenic DAP cells can differentiate into TH+, i.e., functional dopaminergic neurons, in vivo.

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Abstract

本发明公开了一种免疫豁免性诱导性多能干细胞分化的神经细胞及其应用。本发明在诱导性多能干细胞中失活主要组织相容性复合体HLA-I和II类基因后，过表达由CD47和CD24的功能结构域构建的融合蛋白XSG006，获得的诱导性多能干细胞经分化，得到分离的低免疫原性多巴胺能神经前体细胞，可在逃逸T细胞攻击的基础上，进一步逃逸NK细胞及巨噬细胞(Macrophage)的杀伤。并且在体内有效逃逸免疫系统的攻击。在小鼠帕金森氏病模型和非人灵长类(NHP)帕金森氏病模型中能够持续在宿主体内长时间存活并产生活性，从根本上逆转帕金森病。为建立PD治疗策略和设计新型干细胞治疗药物提供实验依据和理论基础。

Description

一种免疫豁免性诱导性多能干细胞分化的神经细胞及其应用
相关申请的交叉引用
本申请要求2023年06月05日向中国国家知识产权局提交的专利申请号为202310657355.7，发明名称为“一种免疫豁免性诱导性多能干细胞分化的神经细胞及其应用”的在先申请的优先权，该件在先申请的全文通过引用的方式结合于本发明中。
技术领域
本发明属于基因工程与干细胞技术交叉领域，具体涉及一种免疫豁免性诱导性多能干细胞分化的神经细胞及其应用。
背景技术
干细胞是一类具备自我更新能力及向特定功能体细胞分化能力的“种子”细胞，依据干细胞特性的程度差异，主要将干细胞分为：全能干细胞(Totipotent stem cells)、多能干细胞(Pluripotent stem cells，PSCs)和成体干细胞(Adult stem cell)。人类胚胎干细胞(hESC)和诱导性多能干细胞(iPSC)具有无限增殖、自我更新和分化到各种类型细胞的潜力，在治疗癌症、神经、心血管等相关疾病方面具有良好的应用前景。
神经科学和再生医学的研究，是国际前沿的重要研究领域；目前大多数老龄化相关的退行性疾病并无实质临床解决方案，巨大的市场需求尚未被满足。在中国迈向老龄化社会的大背景下，神经退行性疾病的研究显得愈发重要。帕金森氏症(PD)是一种严重的神经退行性疾病，其总体发病率仅次于阿尔茨海默氏病(AD)。中脑黑质多巴胺能神经元的进行性丢失是PD发生的主要病理特征。提供细胞移植，让移植的多巴胺能神经元(DAP)替代脑内丢失的多巴胺能神经元而发挥作用，被认为是治疗PD的有效疗法。人多能干细胞可在体外高效分化成为多巴胺神经前体细胞，因此是用于细胞移植疗法治疗帕金森氏症的合适细胞来源。
通过细胞的体外培养或干细胞的诱导分化，能够在体外大量的再生健康的功能细胞，从而通过异体功能细胞移植治疗以帕金森病为代表的重大神经系统疾病，比如脊髓损伤、颅脑外伤等。但是，免疫不相容和移植细胞遭受的免疫排斥仍然是其临床应用的关键障碍。移植自体细胞治疗可以回避免疫排斥问题，但自体iPS分化细胞治疗技术步骤繁琐,造价昂贵,耗费时间,患者需等待较长时间才能获得治疗，而异体iPS分化细胞治疗面临 移植物受到免疫系统排斥问题、长期服用免疫抑制剂对患者将产生严重毒副作用。
因此急需要开发以不依赖于免疫抑制剂的低免疫原性iPS衍生细胞药(即通用型iPS衍生细胞药)疗法，满足再生医学的细胞的需要。
发明内容
针对现有技术所存在的不足，本发明通过敲除诱导性多能干细胞(iPSC)内质网中β-2-微球蛋白(B2M)和敲除MHC-II基因转录的正调节因子CIITA，成功构建B2M/CIITA双等位基因敲除阳性克隆(DKO细胞)；然后使用慢病毒载体在DKO细胞中过表达基于CD47和CD24结构域融合的新基因，由此获得的诱导性多能干细胞可在逃逸T细胞攻击的基础上，进一步逃逸NK细胞的杀伤。该细胞经过分化，得到分离的免疫豁免性多巴胺能神经细胞。所述多巴胺能神经细胞能够显著减少或逃逸免疫系统的识别和攻击，尤其是自然杀伤细胞、巨噬细胞等的攻击，有效地治疗帕金森病。
为了实现上述目的，本发明采用如下技术方案：
第一方面，本发明提供一种从免疫豁免性诱导性多能干细胞(即低免疫原性iPS细胞)分化的分离的低免疫原性多巴胺能神经细胞，包含：
1)HLA-I和HLA-II人类白细胞抗原的表达降低或不表达；
2)表达由CD47和CD24的功能结构域构建的融合蛋白；
所述细胞能逃逸T细胞攻击及NK细胞的杀伤。
在某些方面，所述免疫豁免性诱导性多能干细胞中使用基因编辑工具(诸如TALEN和/或CRISPR系统)靶向编码HLA-I的一种或多种转录调节因子的一个或多个基因，以及编码HLA-II的一种或多种转录调节因子的一个或多个基因，来实现HLA-I和HLA-II基因的表达降低或不表达。
在某些实施方式中，为实现HLA-I和HLA-II基因的表达降低或不表达，所述HLA-I的转录调节因子可优选自：B2M、TAP1、TAP2、TAP相关糖蛋白(Tapasin)或NLRC5中的一个或多个；所述HLA-II的转录调节因子可优选自：CIITA、RFXANK、RFX5、RFXAP中的一个或多个；
根据本发明，所述转录调节因子优选B2M和CIITA。
在某些实施方式中，所述免疫豁免性诱导性多能干细胞还包括通过选择性灭活CIITA基因的稀有切割核酸内切酶靶向CIITA基因的遗传修饰。
在某些实施方式中，所述免疫豁免性诱导性多能干细胞还包括通过选择性灭活B2M 基因的稀有切割核酸内切酶靶向B2M基因的遗传修饰。
在某些实施方式中，其中通过稀有切割内切核酸酶靶向CIITA基因或B2M基因的遗传修饰包括CAS蛋白或编码CAS蛋白的多核苷酸，和至少一个用于特异性靶向CIITA基因或B2M基因的导向核糖核酸序列。
在具体的实施方式中，使用CRISPR/CAS9系统分别对B2M和CIITA外显子区段进行两端直接敲除，其中，针对B2M基因的gRNA的靶序列为SEQ ID NO:2和SEQ ID NO:3，针对CIITA基因的gRNA的靶序列为SEQ ID NO:4和SEQ ID NO:5。
在某些方面，所述免疫豁免性诱导性多能干细胞中通过引入针对编码HLA-I的一种或多种转录调节因子的一个或多个基因，或编码HLA-II的一种或多种转录调节因子的一个或多个基因的基因表达修饰分子，来实现HLA-I和/或HLA-II基因的表达降低或不表达，其中所述基因表达修饰分子包括选自siRNA，shRNA，microRNA，反义RNA和另一种RNA介导的抑制分子中的一种。
在某些方面，所述包含CD47和CD24的功能结构域的融合蛋白中的CD47的功能结构域为CD47跨膜结构域；优选地，所述CD47跨膜结构域的氨基酸序列如SEQ ID NO：6-10任一项所示。
在某些方面，所述包含CD47和CD24的功能结构域的融合蛋白中的CD24的功能结构域为CD24的信号肽序列、CD24成熟肽、CD24胞外肽、CD24膜锚定序列、CD24胞外成熟肽；优选地，所述CD24的功能结构域的氨基酸序列如SEQ ID NO：11-16任一项所示。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47SIRPα结合域和CD24的膜锚定序列连接。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域插入至CD24的胞外肽和膜锚定序列的链接位点。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47链接到CD24的胞外肽后。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24成熟肽链接到CD47的SIRPα结合域后。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24的胞外成熟肽插入到CD47的SIRPα结合域和跨膜结构域链接点。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24 的成熟肽序列和CD47跨膜结构域连接；
优选地，所述由CD47和CD24的功能结构域构建的融合蛋白的氨基酸序列与如SEQ ID NO:1所示的序列具有70％以上的同源性，例如80％以上的同源性，再例如90％以上、95％以上、98％以上的同源性；
进一步优选地，所述由CD47和CD24的功能结构域构建的融合蛋白的氨基酸序列如SEQ ID NO:1所示。
在具体的实施方式中，增加的CD47和CD24的功能结构域构建的融合蛋白表达是在所述的免疫豁免性诱导性多能干细胞中将至少一个拷贝的融合蛋白基因导入所述iPSC中处于启动子的控制下而产生的。
在某些方面，所述编码上述由CD47和CD24的功能结构域构建的融合蛋白核酸序列可以根据所述融合蛋白的氨基酸序列得到。
在某些方面，所述的分离的低免疫原性多巴胺能神经细胞选自多巴胺能神经前体细胞、多巴胺能神经祖细胞、未成熟的多巴胺能神经细胞和成熟的多巴胺能神经细胞；优选为多巴胺能神经前体细胞。
第二方面，本发明提供一种通过体外分化从免疫豁免性诱导性多能干细胞分化的第一方面所述的分离的低免疫原性多巴胺能神经细胞的方法，其中在免疫豁免性iPSC细胞中已消除内源性β-2微球蛋白(B2M)基因活性和内源性II类反式激活因子(CIITA)基因活性；并且表达由CD47和CD24的功能结构域构建的融合蛋白；优选地，所述由CD47和CD24的功能结构域构建的融合蛋白的氨基酸序列与如SEQ ID NO:1所示的序列具有70％以上的同源性，例如80％以上的同源性，再例如90％以上、95％以上、98％以上的同源性；进一步优选地，所述由CD47和CD24的功能结构域构建的融合蛋白的氨基酸序列如SEQ ID NO:1所示；
所述方法包括：
免疫豁免性诱导性多能干细胞分化为低免疫原性多巴胺能神经前体细胞：
(1)免疫豁免性诱导性多能干细胞的培养：
按照约10万每孔的数量将免疫豁免性诱导性多能干细胞以团块的形式在预先包被过Laminin的基质上进行铺板，并使用E8完全培养基进行细胞培养，培养过程中每隔4～5天消化传代；
(2)低免疫原性多巴胺能神经前体细胞的分化：
S1：分化前一天，将汇合度约为70％～80％的免疫豁免性诱导性多能干细胞用 TrypLE酶消化成单细胞，将细胞以8000/cm2的密度接种到预先包被过Vitronectin(Gibco)的6孔板中，接种培养基为StemFit完全培养基，并添加10μM Y27632组分以促进干细胞存活；
S2：细胞贴壁生长24h后，将培养基更换为第一培养基，作为分化的第0天，称为D0，于37℃、5％CO2的二氧化碳细胞培养箱中培养8天，每两天换液一次；
S3：在分化的第9天，用Accutase(Innovative)消化细胞，然后用第二培养基重悬细胞，再将1500万细胞接种至T75悬浮培养瓶中，摇晃均匀后，于37℃、5％CO2的二氧化碳细胞培养箱中培养2天，使细胞在悬浮状态中自发形成神经球，得到中脑底板细胞；
S4：在分化的第11天，将培养基更换为第三培养基，于37℃、5％CO2的二氧化碳细胞培养箱中培养5天，每2.5天换液一次，得到多巴胺能神经祖细胞；
S5：在分化的第16天，将培养基更换为第四培养基，并将500万神经球细胞做贴壁处理，贴于Vitrovectin(Gibco)预先包被的T75培养瓶中继续培养，于37℃、5％CO2的二氧化碳细胞培养箱中培养7天，每2.5天换液一次；
S6：在分化的第23天，用Accutase(Innovative)消化细胞，然后用第五培养基重悬细胞，再将1500万细胞接种至T75悬浮培养瓶中，摇晃均匀后，于37℃、5％CO2的二氧化碳细胞培养箱中培养一天，使细胞在悬浮状态中自发形成神经球，得到多巴胺能神经前体细胞(DAP细胞)；
其中，第一培养基为含有1μM-20μM SB431542、1μM-3μM DMH1、0.2μM-1μM CHIR99021和1μM-1.75μM SAG的神经基础培养基；
优选地，第一培养基为含有10μM SB431542、2μM DMH1、0.6μM CHIR99021和1μM SAG的神经基础培养基；
第二培养基为含有5μM-15μM Y27632、1μM-20μM SB431542、1-3μM DMH1、0.2μM-1μM CHIR99021和1μM-1.75μM SAG的神经基础培养基；
优选地，第二培养基为含有10μM Y27632、10μM SB431542、2μM DMH1、0.6μM CHIR99021和1μM SAG的神经基础培养基；
第三培养基为含有0.8μM-1.2μM DMH1、0.1μM-0.5μM SAG和80ng/mL-120ng/mL成纤维细胞生长因子8b(FGF8b)的神经基础培养基；
优选地，第三培养基为含有1μM DMH1、0.2μM SAG和100ng/mL成纤维细胞生长因子8b(FGF8b)的神经基础培养基；
第四培养基为含有0.1μM-0.5μM SAG和80ng/mL-120ng/mL FGF8b的神经基础培养基；
优选地，第四培养基为含有0.2μM SAG和100ng/mL FGF8b的神经基础培养基；
第五培养基为含有5μM-15μM Y27632、0.1μM-0.5μM SAG和80ng/mL-120ng/mL FGF8b的神经基础培养基；
优选地，第五培养基为含有10μM Y27632、0.2μM SAG和100ng/mL FGF8b的神经基础培养基；
所述神经基础培养基选自IMDM(Iscove's Modified Dulbecco's Medium)培养基、Eagle's Basal Medium(BME)培养基、GMEM培养基、MEM培养基、DMEM培养基，Ham's F-12培养基、RPMI1640培养基、Neurobasal培养基中的至少一种。
优选的，所述神经基础培养基由以下组分组成：98％(v/v)DMEM/F12(Gibco)、1％(v/v)非必须氨基酸(Non-essential amino acid，Gibco)和1％(v/v)N2添加剂(N2 supplement，Gibco)。
第三方面，本发明提供一种组合物，所述组合物包含第一方面所述的分离的低免疫原性多巴胺能神经细胞或根据第二方面的方法制备的分离的低免疫原性多巴胺能神经细胞。
在某些方面，所述组合物包含第一方面所述的分离的低免疫原性多巴胺能神经细胞和一种或多种治疗剂，所述治疗剂包含肽、细胞因子、小分子化合物、大分子、ADC、抗体、纳米粒子、生物类似物、mRNA、中药、蛋白、疫苗、检查点抑制剂、丝裂原、生长因子、小RNA、双链RNA(double stranded RNA；dsRNA)、单核血细胞、饲养细胞、饲养细胞组分或其置换因子、包含一种或多种所关注多核酸的载体、抗体等。
第四方面，本发明提供一种治疗患有神经退行性疾病或病况的患者的方法，所述方法包括施用包含治疗有效量的本发明第一方面所述的分离的低免疫原性多巴胺能神经细胞或根据第二方面的方法制备的分离的低免疫原性多巴胺能神经细胞或第三方面所述的组合物。
在某些方面，所述方法，其中所述组合物还包含治疗有效的载体。
在某些方面，所述方法，其中所述分离的低免疫性多巴胺能神经细胞在可生物降解的支架上。
在某些方面，所述方法，其中所述施用包括移植或注射。
在某些方面，所述方法，其中所述神经退行性疾病或病况选自帕金森氏病、亨廷顿 氏病和多发性硬化症。
第五方面，本发明提供第一方面所述的分离的低免疫原性多巴胺能神经细胞或根据第二方面的方法制备的分离的低免疫原性多巴胺能神经细胞或第三方面所述的组合物在制备用于神经退行性疾病或病况治疗的产品中的应用。
在某些方面，所述应用，其中所述神经退行性疾病或病况选自帕金森氏病、亨廷顿氏病和多发性硬化症；优选为帕金森氏病。
本发明的有益效果：
1.本发明在诱导性多能干细胞中失活主要组织相容性复合体MHC-I和II类基因后，过表达由CD47和CD24的功能结构域构建的融合蛋白XSG006，获得的诱导性多能干细胞经分化，得到分离的低免疫原性多巴胺能神经前体细胞，可在逃逸T细胞攻击的基础上，进一步逃逸NK细胞及巨噬细胞(Macrophage)的杀伤。
2.本发明分离的低免疫原性多巴胺能神经前体细胞可在体内(人源化小鼠及非人灵长类)有效逃逸免疫系统的攻击。在小鼠帕金森氏病模型中，进行本发明分离的低免疫原性多巴胺能神经前体细胞移植，4个月后检测结果显示在阿扑吗啡(APO)诱导的旋转实验中小鼠旋转次数有显著降低，且具有统计学意义；在圆筒实验中小鼠右前肢碰壁次数明显提高，基本恢复至造模前水平，且具有统计学意义；在转棒实验中，小鼠在转棒上停留的时间明显提高，且具有统计学意义。在脑组织冰冻切片免疫荧光染色结果中，移植细胞大量存活于移植部位且一定数量分化为多巴胺能神经元。移植DAP注射液可明显改善小鼠帕金森病行为并可从根本上促进了纹状体内的移植细胞分化生成多巴胺能神经元以从根本上逆转帕金森病。
3.在非人灵长类(NHP)帕金森氏病模型中，本发明分离的猴低免疫原性多巴胺能神经前体细胞能够持续在宿主体内长时间存活并产生活性。
4.对具有完全免疫系统的非人灵长类(NHP)进行同种异体DAP细胞移植，相对于猴WT-DAP细胞在猴体内存活期短、、很快因出现排异反应而消失，猴DKO+G6-DAP细胞具有低免性可逃逸免疫系统攻击，在具有完全免疫系统的同种异体猴脑内长期存活。体内安全性检测显示低免疫原性多巴胺能神经前体细胞(DKO+G6-DAP)与未编辑细胞(WT-DAP)相比具有无差异的增殖能力，且均含有极少量的Ki67，提示体内致瘤风险低。而且，低免疫原性DAP细胞可在体内分化生成TH+即有功能的多巴胺能神经元。
附图说明
图1.B2M的CRISPR/CAS9基因敲除策略示意图。
图2.CIITA的CRISPR/CAS9基因敲除策略示意图。
图3.pGC-EF1a质粒的结构示意图。
图4.实施例1中使用流式细胞计数仪检测各DAP细胞中OCT-4表达图；
图5.实施例1中使用流式细胞计数仪检测各DAP细胞中FOXA2、PAX6、OTX2、和LMX1A表达图；
图6.实施例1中使用免疫荧光检测各DAP细胞中OTX2、EN1、LMX1A和FOXA2的表达图；
图7.实施例1中用INF-gamma刺激各DAP细胞，通过流式细胞计数仪检测各种细胞中HLA-I/II的表达图；
图8.实施例1中对构建的DKO+G6细胞株及其衍生的DAP细胞进行序列rt-PCR鉴定。
其中，T细胞为检测HLA-I/II类分子的阳性对照；
图9.实施例2中使用RTCA检测PBNK细胞对WT、DKO及DKO+G6分化的DAP细胞的杀伤结果图；
图10.实施例2中使用MAC对WT、DKO及DKO+G6分化的DAP细胞杀伤的照片；
图11.实施例2中使用RTCA检测MAC细胞对WT、DKO及DKO+G6分化的DAP细胞的杀伤结果图；
图12.实施例2中DKO+G6分化的DAP细胞在人源免疫系统小鼠中的免疫逃逸作用验证结果图；
图13.实施例3中PD小鼠模型移植WT-DAP注射液后阿扑吗啡(APO)诱导旋转实验结果；
图14.实施例3中PD小鼠模型移植WT-DAP注射液后圆筒实验结果；
图15.实施例3中PD小鼠模型移植WT-DAP注射液后转棒实验结果；
图16.实施例3中细胞移植4个月的PD小鼠脑切片免疫荧光染色结果；
其中16B是16A中截取部分的4x、20x、60x放大。
图17.实施例4中PD猴模型建模成功后PET-CT结果展示图。
图18.实施例4中使用免疫荧光检测各猴干细胞衍生的DAP细胞中OTX2、EN1、LMX1A和FOXA2的表达图；
图19.实施例4中WT-DAP(左脑)和DKO+G6-DAP(右脑)细胞移植前后猴脑部MRI结果图；
图20.实施例4中WT-DAP和DKO+G6-DAP细胞移植前后PD猴左右收采食率统计图；
图21.实施例5中WT-DAP和DKO+G6-DAP细胞移植示意图及移植前后猴脑部MRI结果图(A)；MRI检测细胞大小统计图(B)。
图22.实施例5中小鼠脑免疫荧光染色(A)，hNA(红)&Ki67(绿)&DAPI(蓝)共染，其中hNA染移植细胞，Ki67标记增殖细胞；免疫荧光染色统计(B)。
图23：实施例5中DKO+G6-DAP细胞移植PD-NOG后脑部免疫荧光染色结果。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。
除非另外定义或由背景清楚指示，否则在本公开中的全部技术与科学术语具有如本公开所属领域的普通技术人员通常理解的相同含义。
免疫豁免性多能干细胞的详细描述、其产生方法及其使用方法见于2022年7月08日提交的202210806871.7和2023年3月24日提交的PCT/CN2023/083761中，包括序列表、附图和实例的公开内容以引用的方式整体并入本文。
定义
术语“多能细胞”是指可以自我更新并增殖同时保持未分化状态并且可以在适当条件下被诱导以分化为专门的细胞类型的细胞。如本文所用，术语“多能细胞”涵盖胚胎干细胞和其他类型的干细胞，包括胎儿、羊膜或体细胞干细胞。示例性的人干细胞系包括H9人胚胎干细胞系。其他示例性干细胞系包括可通过National Institutes of Health Human Embryonic Stem Cell Registry和Howard Hughes Medical Institute HUES collection获得的那些(描述于Cowan,C.A.等人,New England J.Med.350:13.(2004)，其通过引用 整体并入本文)
如本文所用，“多能干细胞”具有分化成三个胚层中任一个的潜力：内胚层(例如，胃连接、胃肠道、肺等)、中胚层(例如，肌肉、骨骼、血液，泌尿生殖组织等)或外胚层(例如表皮组织和神经系统组织)。如本文所用，术语“多能干细胞”还涵盖“诱导多能干细胞”或“iPSC”，其是来源于非多能细胞的一种多能干细胞。亲代细胞的例子包括已经通过各种方式被重新编程以诱导多能的未分化表型的体细胞。这样的“iPS”或“iPSC”细胞可以通过诱导某些调节基因的表达或通过外源施加某些蛋白质来产生。诱导iPS细胞的方法是本领域已知的，并在下文进一步描述。(参见例如，Zhou等人,Stem Cells27(11):2667-74(2009)；Huangfu等人,Nature Biotechnol.26(7):795(2008)；Woltjen等人,Nature 458(7239):766-770(2009)；和Zhou等人,Cell Stem Cell 8:381-384(2009)；每篇文献均通过引用整体并入本文)。
如本文所用，术语“受试者”或“患者”是指任何动物，例如家养动物、动物园动物或人。“受试者”或“患者”可以是哺乳动物，如狗、猫、鸟、家畜或人。“受试者”和“患者”的具体例子包括但不限于患有与肝脏、心脏、肺、肾脏、胰腺、脑、神经组织、血液、骨骼、骨髓等相关的疾病或病症的个体(特别是人)。
在本文中，“免疫豁免性诱导性多能干细胞”是指保留其多能特征并且当转移到同种异体宿主中时引起减少的免疫排斥反应的多能细胞。在优选的实施方案中，所述细胞不引起免疫反应。因此，“免疫豁免性”是指与本文概述的免疫工程化之前的亲代(即“野生型”或“wt”)细胞的免疫反应相比，显著降低或消除的免疫反应。在许多情况下，该细胞在免疫学上是低免疫原性的，但仍保留了多能性及分化潜能。
“HLA”或“人白细胞抗原”复合物是编码人中主要组织相容性复合物(MHC)蛋白的基因复合物。构成HLA复合物的这些细胞表面蛋白负责调节对抗原的免疫反应。在人中，有两种MHC，即I类和II类，“HLA-I”和“HLA-II”。HLA-I包括三种蛋白质，即HLA-A、HLA-B和HLAC，它们从细胞内部呈递肽，而HLA-I复合物呈递的抗原吸引杀伤性T细胞(也称为CD8+T-细胞或细胞毒性T细胞)。HLA-I蛋白与β-2微球蛋白(B2M)相关。HLA-II包括五种蛋白质，即HLA-DP、HLA-DM、HLA-DOB、HLA-DQ和HLA-DR，它们将抗原从细胞外呈递给T淋巴细胞。这刺激CD4+细胞(也称为T辅助细胞)。应当理解，“MHC”或“HLA”的使用并不意味着是限制性的，因为它取决于基因是来自人(HLA)还是鼠(MHC)。因此，由于它涉及哺乳动物细胞，这些术语在本文中可以互换使用。
本文中“CD47”又称为整合素相关蛋白(integrin-associated protein，IAP)，广泛的表达于各种细胞的表面，可与信号调节蛋白α(SIRPα)、血小板反应蛋白-1(TSP-1)相互作用，在介导某些心血管疾病的氧化应激反应及免疫修复过程发挥着重要的作用。CD47作为膜蛋白免疫球蛋白超家族的成员，含有3个组分：氨基端胞外免疫球蛋白可变结构域(IgV)样结构域、5个结构域跨膜区域和羧基末端剪接变异细胞质尾部。
本文中“CD24”全称为cluster of differentiation 24，又称热稳定抗原，是一种高度糖基化的糖基磷脂酰肌醇锚定表面蛋白，已知与先天免疫细胞上的Siglec-10(sialic-acid-binding Ig-like lectin10)相互作用，以抑制响应感染、败血症、肝损伤和慢性移植物抗宿主疾病的破坏性炎症反应，CD24是一种小分子量的唾液酸糖蛋白，由31个氨基酸组成，具有典型的黏蛋白结构，通过其糖基化酰肌醇(GPI)锚定定位于脂筏中。
本文的“基因敲除”是指使特定基因在其所驻留的宿主细胞中失活的过程，导致不产生感兴趣的蛋白质或形成失活形式。如本领域技术人员将理解的并且在下文中进一步描述，这可以通过许多不同的方式来完成，包括从基因中去除核酸序列，或用其他序列中断序列，改变阅读框，或改变核酸的调节组分。例如，感兴趣的基因的编码区的全部或部分可以被去除或被“无义”序列替代，调节序列如启动子的全部或一部分可以被去除或替代，翻译起始序列可以被去除或替代等。
本文中的“基因敲入”是指向宿主细胞增加遗传功能的过程。这导致编码的蛋白的水平增加。如本领域技术人员将理解的，这可以通过几种方式来实现，包括将基因的一个或多个其他拷贝添加至宿主细胞或改变内源基因的调节组分，从而增加蛋白质的表达。这可以通过修饰启动子、添加不同的启动子、添加增强子或修饰其他基因表达序列来实现。
本文中的“同种异体”是指宿主生物和细胞移植物的遗传不相似性，其中产生免疫反应。
在两个或更多个核酸或多肽序列的上下文中，术语“同一性”百分比是指当就最大对应性进行比较和比对时具有指定百分比的相同核苷酸或氨基酸残基的两个或更多个序列或子序列，如使用下文描述的序列比较算法之一(例如，BLASTP和BLASTN或本领域技术人员可用的其他算法)或通过目视检查测量的。取决于应用，“同一性”百分比可以存在于被比较的序列的区域上，例如存在于功能结构域上，或者可替代地存在于要被比较的两个序列的全长上。对于序列比较，通常将一个序列用作与测试序列进行比较的参考序列。使用序列比较算法时，将测试序列和参考序列输入计算机，必要时指定子 序列坐标，并指定序列算法程序参数。然后，序列比较算法基于指定的程序参数计算测试序列相对于参考序列的序列同一性百分比。
用于比较的序列的最佳比对可以例如通过Smith&Waterman,Adv.Appl.Math.2:482(1981)的局部同源性算法，通过Needleman&Wunsch,J.Mol.Biol.48:443(1970)的同源性比对算法，通过Pearson&Lipman,Proc.Natl.Acad.Sci.USA 85:2444(1988)的相似性搜寻方法，通过这些算法的计算机实施(Wisconsin Genetics Software Package,Gen etics Computer Group,575Science Dr.,Madison,Wis.中的GAP、BESTFIT、FASTA和TFASTA)或通过目视检查(一般参见Ausubel等人，见下文)来进行。
BLAST算法是适合确定序列同一性和序列相似性百分数的算法的一个例子，其描述于Altschul等人,J.Mol.Biol.215:403-410(1990)。可通过美国国家生物技术信息中心(www.ncbi.nlm.nih.gov/)公开获得用于进行BLAST分析的软件。
“同源物”是在核苷酸序列、肽序列、功能或结构水平上与参考分子相似的生物活性分子。同源物可以包括与参考序列具有一定百分比同一性的序列衍生物。因此，在一个实施方案中，同源或衍生序列共有至少70％的序列同一性。在一个具体的实施方案中，同源或衍生序列共有至少80％或85％的序列同一性。在一个具体的实施方案中，同源或衍生序列共有至少90％的序列同一性。在一个具体的实施方案中，同源或衍生序列共有至少95％的序列同一性。在一个更具体的实施方案中，同源或衍生序列共有至少50、55、60、65、70、75、85、86、87、88、89、90、91、92、93、94、95、96，97、98或99％的序列同一性。同源或衍生核酸序列也可以通过它们在高严格性杂交条件下保持与参考核酸序列结合的能力来定义。与参考分子具有结构或功能相似性的同源物可以是参考分子的化学衍生物。检测、产生和筛选结构和功能同源物以及衍生物的方法是本领域已知的。
本文中的“多巴胺能神经前体细胞”通常是指能够在体外或体内增殖和/或分化为多巴胺能神经元的细胞。多巴胺能神经前体细胞可以来自中脑腹侧神经细胞，可以由多能干细胞分化而来。多巴胺能神经前体细胞也可以从其他细胞类型分化或重编程。
本文中的“多巴胺能神经细胞”是选自多巴胺能神经前体细胞、多巴胺能神经祖细胞、未成熟的多巴胺能神经细胞和成熟的多巴胺能神经细胞；优选为多巴胺能神经前体细胞。
实施例1.利用低免疫原性干细胞(即低免疫原性iPS细胞)分化为中脑多巴胺能神经前体(DAP)细胞
A、低免疫原性干细胞制备方法以及干细胞体外分化多巴胺能神经前体细胞的方法
将WT、DKO及DKO+G6细胞传代后铺至已预包被Matrigel的培养瓶中，培养24h后换至预分化培养基，诱导细胞向中脑细胞分化；在分化9d后，可获得高纯度的中脑细胞。其后，通过加入神经祖细胞培养基对获得的中脑细胞进行扩增，可获得大量的高纯度的神经祖细胞；最后，再通过加入神经前体细胞培养基，神经祖细胞进一步分化成为神经细胞。该细胞在体外可分化成为多巴胺能神经前体(DAP)细胞。
1.低免疫原性干细胞制备方法
1.1细胞培养试剂：
1.2 DKO细胞制备方法：
本发明选用了人诱导性多能干细胞(iPSC细胞，人血液PBMC通过CTSTM CytoTuneTM-iPS2.1仙台病毒重编程试剂盒(货号:A34546)重编程为iPSC细胞，即WT细胞)，使用CRISPR/CAS9敲除内质网中β-2-微球蛋白(B2M)，使细胞表面HLA-I不能形成功能性分子，从而逃逸同种异体CD8+T细胞的杀伤；逃逸CD4+T细胞的杀伤则是通过敲除HLA-II基因转录的正调节因子CIITA，而降低HLA-II类分子表达。
其中，B2M的CRISPR/CAS9基因敲除策略如图1所示，使用B2M-gRNA1和B2M-gRNA2对B2M外显子区段进行两端直接敲除，然后分别使用B2M-F1/R1和B2M-F2/R2两对PCR引物进行基因组序列敲除验证。
gRNA序列:
B2M-gRNA1：CGTGAGTAAACCTGAATCTT
B2M-gRNA2：AGTCACATGGTTCACACGGC
鉴定引物
B2M-F1:TGGGGCCAAATCATGTAGACTC
B2M-R1:TCAGTGGGGGTGAATTCAGTGT
B2M-F2+B2M-R2＝608bp
敲除后：无条带
B2M-F2:CAGAAGTCCTTGAGAGCCTCC
B2M-R2:TGTGCATCAGTATCTCAGCAGG
B2M-F2+B2M-R2＝812bp
敲除后：569bp。
另外，CIITA的CRISPR/CAS9基因敲除策略如图2所示，使用CIITA-gRNA1和CIITA-gRNA2对CIITA外显子区段进行两端直接敲除，然后分别使用CIITA-F1/R1和CIITA-F2/R2两对PCR引物进行基因组序列敲除验证。
gRNA序列:
CIITA-gRNA1:GATATTGGCATAAGCCTCCC
CIITA-gRNA2:CATCGCTGTTAAGAAGCTCC
鉴定引物：
CIITA-F1:CTGTGCCTCTACCACTTCTATG
CIITA-R1:CCTTCCATGTCACACAACAGCC
CIITA-F1+CIITA-R1＝368bp
敲除后：无条带
CIITA-F2:TGGAATCCACACTTTCCAGTTC
CIITA-R2:TGGAGTCTCCGTTCCTCCAG
CIITA-F2+CIITA-R2＝889bp
敲除后：459bp
具体操作如下：
1)在Matrigel包被的6孔板上正常使用mTeSR1培养人类诱导性多能干细胞至80％密度。使用TRYPLE消化后加入DMEM/F12中和，计数。吸取2×106细胞于EP管中，离心后弃上清。
2)根据Neon转染系统100μL电转体系，加入15μg TrueCutTM Cas9 Protein+3μg gRNA(B2MgRNA1+B2MgRNA2+CIITA gRNA1+CIITA gRNA2)组成RNP体系，混匀室温下放置20min。
3)100μL RNP电转体系重悬细胞，Neon转染系统进行电转，电转参数为1200V，30ms，1pause。电转后细胞迅速加入提前预热的培养基，均匀接种于1孔包被了Matrigel的6孔板中。
4)每天更换新鲜mTeSR1培养基。待单细胞生长起来，挑取单个克隆于48孔板内，待克隆扩增后，收取基因组样品进行PCR检测基因编辑情况，PCR阳性克隆送公司做Sanger测序进一步验证。
5)鉴定阳性的B2M/CIITA双等位基因敲除克隆DKO细胞扩增培养及冻存。
6)使用下列B2M-F/R和CIITA-F/R引物进行qPCR检测B2M/CIITA双等位基因敲除克隆DKO的B2M和CIITA在RNA水平的表达情况，确定敲除。
B2M-F:AAGATGAGTATGCCTGCCGT
B2M-R:ATGCGGCATCTTCAAACCTC
CIITA-F:CCTGGAGCTTCTTAACAGCGA
CIITA-R:TGTGTCGGGTTCTGAGTAGAG。
1.3 DKO+G6制备方法
在1.2制备的DKO细胞中通过慢病毒感染的方式进行过表达融合蛋白XSG006，形成融合蛋白稳转细胞株DKO+G6。融合蛋白XSG006由CD47和CD24的功能结构域构建的融合蛋白是将CD24的成熟肽序列和CD47跨膜结构域连接，其氨基酸序列如SEQ ID NO.1所示。MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNSGLAPNPT NATTKAAGGGGGSGGGGSGGGGSNILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFV，直接合成编码XSG006(XSG006的氨基酸序列如SEQ ID NO：1所示)的核酸序列并构建在由EF1a启动，且带puromycin筛选标记的慢病毒质粒中(pGC-EF1a)，pGC-EF1a质粒的结构如图3所示。质粒用BamHI/NheI酶切，连接成功后采用Sanger测序验证插入序列正确性并进行病毒包装。对1.2获得的DKO人类诱导性多能干细胞进行转染后换成带purom ycin的培养基进行筛选。XSG006为完全外源序列，构建的DKO+G6细胞使用PCR检测序列在基因组中的插入情况，DKO细胞为阴性对照，确认表达无误后进行细胞扩增和后续 功能性检测。
XSG006 F1：CCAGATCTACAGCAGCGAGA
XSG006 R1：GTTCTTCAGGCTGTACTCGC
XSG006 F+XSG006 R＝352bp
XSG006 F2：CCAGATCTACAGCAGCGAGA
XSG006 R2：CCAGGATGTAGGCGATCACC
XSG006 F+XSG006 R＝488bp
2.干细胞体外分化多巴胺能神经前体细胞的方法
2.1人诱导性多能干细胞(WT细胞)、DKO细胞及DKO+G6细胞分化为多巴胺能神经前体细胞的试剂盒，包含：第一培养基、第二培养基、第三培养基、第四培养基和第五培养基；
其中，第一培养基为含有10μM SB431542、2μM DMH1、0.6μM CHIR99021和1μM SAG的神经基础培养基；
第二培养基为含有10μM Y27632、10μM SB431542、2μM DMH1、0.6μM CHIR99021和1μM SAG的神经基础培养基；
第三培养基为含有1μM DMH1、0.2μM SAG和100ng/mL成纤维细胞生长因子8b(FGF8b)的神经基础培养基；
第四培养基为含有0.2μM SAG和100ng/mL FGF8b的神经基础培养基；
第五培养基为含有10μM Y27632、0.2μM SAG和100ng/mL FGF8b的神经基础培养基；
其中，神经基础培养基由以下组分组成：98％(v/v)DMEM/F12(Gibco)、1％(v/v)非必须氨基酸(Non-essential amino acid，Gibco)和1％(v/v)N2添加剂(N2 supplement，Gibco)。
2.2人诱导性多能干细胞(WT细胞)、DKO细胞及DKO+G6细胞分化为多巴胺能神经前体细胞的方法，包含采用2.1的试剂盒的步骤，具体如下：
人诱导性多能干细胞分化为多巴胺能神经前体细胞：
1)人多能干细胞的培养：
按照约10万每孔的数量将人诱导性多能干细胞，DKO细胞或DKO+G6细胞以团块的形式在预先包被过Laminin的基质上进行铺板，并使用E8完全培养基进行细胞培养，培养过程中每隔4～5天消化传代；
2)多巴胺能神经前体细胞的分化：
S1：分化前一天，将汇合度约为70％～80％的人多能干细胞，DKO细胞及DKO+G6 细胞用TrypLE酶消化成单细胞，将细胞以8000/cm2的密度接种到预先包被过Vitronectin(Gibco)的6孔板中，接种培养基为StemFit完全培养基，并添加10μM Y27632组分以促进干细胞存活；
S2：细胞贴壁生长24h后，将培养基更换为2.1的试剂盒中的第一培养基(作为分化的第0天，D0)，于37℃、5％CO2的二氧化碳细胞培养箱中培养8天(每两天换液一次)；
S3：在分化的第9天，用Accutase(Innovative)消化细胞，然后用2.1的试剂盒中的第二培养基重悬细胞，再将1500万细胞接种至T75悬浮培养瓶中，摇晃均匀后，于37℃、5％CO2的二氧化碳细胞培养箱中培养2天，使细胞在悬浮状态中自发形成神经球，得到中脑底板细胞；
S4：在分化的第11天，将培养基更换为2.1中的试剂盒中的第三培养基，于37℃、5％CO2的二氧化碳细胞培养箱中培养5天(每2.5天换液一次)，得到多巴胺能神经祖细胞；
S5：在分化的第16天，将培养基更换为2.1中的试剂盒中的第四培养基，并将500万神经球细胞做贴壁处理(贴于Vitrovectin(Gibco)预先包被的T75培养瓶中继续培养)，于37℃、5％CO2的二氧化碳细胞培养箱中培养7天(每2.5天换液一次)；
S6：在分化的第23天，用Accutase(Innovative)消化细胞，然后用2.1的试剂盒中的第五培养基重悬细胞，再将1500万细胞接种至T75悬浮培养瓶中，摇晃均匀后，于37℃、5％CO2的二氧化碳细胞培养箱中培养一天，使细胞在悬浮状态中自发形成神经球，得到多巴胺能神经前体细胞(DAP细胞)，其中由iPS细胞分化得到的DAP细胞命名为WT-DAP,由DKO细胞分化得到DAP细胞命名为DKO-DAP细胞，由DKO+G6分化得到的DAP细胞命名为DKO+G6-DAP细胞；
B、低免疫原性干细胞分化多巴胺能神经前体(DAP)细胞特性的分析
1.确认低免疫原性干细胞衍生的DAP细胞标志物
使用流式细胞术及免疫荧光对衍生细胞的身份进行确认。首先验证WT、DKO及DKO+G6分化的DAP细胞中干性基因OCT4的表达均<1％(图4)，确认其已退出多能性且完全为分化细胞。其次通过FACS检测WT、DKO及DKO+G6分化的DAP细胞中多巴胺能神经前体细胞标志基因FOXA2、PAX6、OTX2、和LMX1A的表达量(图5)。
同时还用免疫荧光检测WT、DKO及DKO+G6的衍生细胞表达中多巴胺能神经前体细胞标志基因。具体操作为将细胞铺板在12孔板中，待细胞长到60-80％的密度后吸去培养基，加入4％多聚甲醛进行固定。细胞破膜后使用OTX2、EN1、LMX1A和FOXA2的一抗在4℃过夜孵育，洗去一抗后室温孵育带有荧光标记的二抗，随后使用荧光显微镜进 行拍照。结果如图6所示，所有细胞均表达多巴胺能神经前体细胞标志基因。
2.低免疫原性衍生细胞低免疫原性表型保留的测定
使用INF-gamma刺激WT及DKO+G6分化的DAP细胞：细胞铺于孔板中，第二天换液时将含有INF-gamma的培养基加入到细胞中。待48h后将细胞消化，使用流式检测HLA-I/II的表达情况。结果如图7所示，B2M/CIITA双等位基因敲除的DKO+G6干细胞阳性克隆分化为DAP后依旧不表达HLA-I/II，且不能响应INF-gamma的刺激而上调HLA-I/II。
XSG006的分子序列为完全外源序列，使用rt-PCR比较WT与由其分化的多巴胺神经前体细胞以及DKO+G6与由其分化的多巴胺神经前体细胞之间的XSG006表达的稳定性(图8)。WT-DAP、DKO-DAP细胞为阴性对照，确认表达无误后进行细胞扩增和后续功能性检测。
使用引物进行XSG006过表达确认
XSG006 F1：CCAGATCTACAGCAGCGAGA
XSG006 R1：GTTCTTCAGGCTGTACTCGC
XSG006 F+XSG006 R＝352bp
C、结论
低免疫原性干细胞可在体外成功分化为多巴胺能神经前体(DAP)细胞，衍生细胞具有DAP细胞特性，且保留了低免分子的表达。
实施例2.低免疫原性多巴胺能神经前体细胞低免疫原性验证
A、背景：
本发明选用的人诱导性多能干细胞使用CRISPR/CAS9敲除内质网中β-2-微球蛋白(B2M)，使细胞表面HLA-I不能形成功能性分子，从而逃逸同种异体CD8+T细胞的杀伤；逃逸CD4+T细胞的杀伤则是通过敲除HLA-II基因转录的正调节因子CIITA，而降低HLA-II类分子表达。并通过慢病毒的方式在B2M/CIITA双等位基因敲除的细胞(DKO)中表达XSG006以逃避固有免疫的杀伤。成功构建的细胞为DKO+G6，为低免疫原性干细胞。由实施例1的低免疫原性干细胞衍生而来的多巴胺能神经前体细胞(DKO+G6-DAP)应同样具有不激活免疫系统并逃逸其杀伤的能力。
B、体外检测逃逸免疫系统的杀伤
1.逃逸T+NK混合淋巴细胞(PBNK)杀伤功能验证
提前在PBMC中添加NK激活因子，提高PBMC中的NK比例以及T细胞的杀伤性能，将活化的混合淋巴细胞(PBNK)作为效应细胞进行RTCA实验，综合评判DKO+G6-DAP细胞的免疫逃逸能力(PMID:33309274)。具体操作为在XCelligence平台(ACEA BioSciences，San Diego，CA.)上进行PBNK细胞杀伤测定。将WT-DAP、DKO-DAP和DKO+G6-DAP细胞重悬于100μl细胞特异性培养基(NeurobasalTM培养基,货号:21103049，Thermofisher)中，并铺于包被有Matrigel(Sigma-Aldrich)的96孔E-板(ACEA BioSciences)上。在细胞指标值达到1之后，以1∶1的E∶T比添加T细胞。用RTCA软件(ACEA)对数据进行标准化和分析。结果如图9所示，仅DKO+G6-DAP细胞可逃逸PBNK的杀伤。
2.逃逸巨噬细胞(MAC)杀伤检测
将WT、DKO及DKO+G6分化的DAP细胞铺于12孔板，24h后弃除培养基并加入MAC细胞进行共培养。24h观察去除MAC细胞后剩余的WT、DKO及DKO+G6分化的DAP细胞，结果如图10所示。
在XCelligence平台(ACEA BioSciences，San Diego，CA.)上进行MAC细胞杀伤测定。将WT-DAP、DKO-DAP和DKO+G6-DAP细胞重悬于100μl细胞特异性培养基中，并铺于包被有Matrigel(Sigma-Aldrich)的96孔E-板(ACEA BioSciences)上。在细胞指标值达到1之后，以1∶1的E∶T比添加MAC细胞。用RTCA软件(ACEA)对数据进行标准化和分析。结果如图11所示，两个实验结果均说明，相较DKO分化的DAP细胞，WT及DKO+G6分化的DAP细胞被MAC细胞杀伤的更少，说明WT和DKO+G6分化的DAP细胞可逃逸MAC细胞杀伤的能力显著高于DKO分化的DAP细胞。
C、使用人源化小鼠检测低免疫原性DAP细胞在体内的存活情况
通过观察人源化小鼠脑部注射生物学发光检测(BLI)来观察DAP细胞在体内的存活情况。具体操作为：将携带荧光素酶(luc)的慢病毒感染WT和DKO+G6分化的DAP细胞，然后将表达luc的细胞移植到CD34+HSC重建的人源免疫系统小鼠的脑内。通过腹腔注射luc的发光底物D-荧光素(A025011,上海翊圣生物科技有限公司)，并使用活体成像仪(iVIS spectrum,PerkinElmer)检测细胞的荧光强度，即可指示细胞在小鼠体内的存活情况。持续检测移植部位荧光51天，WT分化的DAP细胞荧光值趋于背景值，而DKO+G6分化 DAP细胞移植后可以检测到持续上升的荧光值，说明DKO+G6分化的DAP细胞相比于WT分化的DAP细胞，可在体内有效逃逸免疫系统的攻击(图12)。
实施例3.小鼠帕金森氏病模型中的多巴胺能神经前体细胞
此实施例描述了一项在小鼠模型上探究来自干细胞的体外分化的多巴胺能神经前体细胞(DAP)用于治疗帕金森氏病的用途的研究。采用scid beige小鼠进行造模，通过阿扑吗啡诱导的旋转、圆筒实验和转棒实验三种行为学进行检测，选出符合标准的造模小鼠进行细胞移植。实验设立溶媒(细胞保存液，人工脑脊液ACSF，懋康生物，MX0951)对照组和受试物组(WT-DAP注射液和DKO+G6-DAP注射液)，给药后通过脑组织病理切片免疫荧光染色及三种行为学检测三组小鼠的行为，比较三组小鼠的行为学差异、给药前后小鼠自身的行为学差异，以及给药部位细胞留存和多巴胺能神经元的变化。
A.构建帕金森病(Parkinson’s disease，PD)小鼠模型
用异氟烷气麻机麻醉scid beige小鼠，将麻醉好的小鼠固定在小鼠脑立体定位仪上，分别立体定位缓慢注射6-羟基多巴胺(6-OHDA)溶液(H4381-100mg(sigma))进小鼠左脑黑质中。术后进行圆筒实验，转棒实验和阿扑吗啡诱导旋转的行为学检测，确定成模小鼠。
B.对帕金森病(Parkinson’s disease，PD)小鼠进行DAP细胞移植
对成模小鼠进行分组注射，分别立体定位缓慢注射2μL WT-DAP注射液、2μLDKO+G6-DAP注射液和2μL细胞保存液进小鼠脑部的左纹状体。术后每月进行一次行为学检测，持续4个月。最后一次检测后对小鼠进行脑部取材。
C.治疗后帕金森病(Parkinson’s disease，PD)小鼠行为学检测
1.阿扑吗啡(APO)诱导旋转实验
从APO诱导的旋转实验结果来看，相较于移植前，4个月时受试物组小鼠旋转次数 显著降低；说明WT-DAP注射液后改善了模型小鼠的偏瘫性旋转行为(图13)。
2.小鼠圆筒实验结果
从圆筒实验结果来看，相较于移植前，4个月时受试物组小鼠有显著行为学改善(p<0.05)，右前肢碰壁次数所占比例明显提高，说明移植WT-DAP注射液后对改善模型小鼠的帕金森相关静止性震颤行为学有效(图14)。
3.小鼠转棒实验结果
从转棒实验结果来看，相较于移植前，4个月时受试物组小鼠有显著的行为学改善(p<0.05)，在转棒上停留的时间明显提高；相较于对照组，行为学改善也比较显著(p<0.05)。说明移植WT-DAP注射液后对改善模型小鼠的姿势步态障碍行为有效(图15)。
D.PD小鼠脑部切片染色
PD小鼠脑组织冰冻切片免疫荧光及免疫组化染色：组织切片使用PBS漂洗5min，3次；封闭1h，37℃；封闭液为含5％BSA、0.3％triton-100的PBS溶液。加一抗，24h～48h，4℃冰箱湿盒中缓慢反应；一抗稀释液为含1％BSA、0.3％triton-100的PBS溶液。PBS漂洗5min，3次后，荧光二抗室温避光孵育1～2h。然后PBS漂洗5min，3次，滴加DAPI封片后封片，显微镜下拍摄。对细胞移植4个月后受试物组小鼠的脑组织冠状冰冻切片进行stem101(红)&TH(绿)&DAPI(蓝)的共染(图16)。染色结果显示，移植部位分化生成多巴胺能神经元。
本实施例设立溶媒对照组(细胞保存液)和受试物组(WT-DAP注射液和DKO+G6-DAP注射液)，实验后通过三种行为学检测三组小鼠的行为，比较三组小鼠的行为学差异及实验前后小鼠自身的行为学差异。4个月后检测结果显示在阿扑吗啡(APO)诱导的旋转实验中两组受试物组小鼠旋转次数有显著降低，且具有统计学意义；在圆筒实验中两组受试物组小鼠右前肢碰壁次数明显提高，基本恢复至造模前水平，且具有统计学意义；在转棒实验中，两组受试物组小鼠在转棒上停留的时间明显提高，且具有统计学意义。在脑组织冰冻切片免疫荧光染色结果中，移植细胞大量存活于移植部位且一定数量分化为了多巴胺能神经元。移植DAP注射液可明显改善小鼠帕金森病行为并可从根本上促进了纹状体内的移植细胞分化生成多巴胺能神经元以从根本上逆转帕金森病。
实施例4.非人灵长类(NHP)帕金森氏病模型中的低免疫原性多巴胺能神经前体细胞
此实施例描述了一项在非人灵长类(NHP)模型上探究来自猴干细胞(iPSC)的体外 分化的多巴胺能神经前体细胞(DAP)用于治疗帕金森氏病的用途的研究。采用注射MPTP对非人灵长类(Non human primate,NHP)进行造模，使用用于评价PD症状和所移植细胞的功能的技术和方法定期观测并评价动物。在一些情况下，所述方法包括MRI扫描和分析、PET分析、视频分析和行为分析等(参见例如Kikuchi等人，Nature，2017，548，592-596)。
A.构建帕金森病(Parkinson’s disease，PD)非人灵长类(NHP)模型
将猴每周用MPTP盐酸盐静脉内注射两次，直至其存在帕金森症状，诸如但不限于震颤、运动徐缓和平衡受损。并使用18F-PECNT对多巴胺转运体进行特异性靶向标记，PET-CT结果显示，造模后NHP左右脑的多巴胺能神经细胞得到了有效清除(图17)，说明帕金森病(Parkinson’s disease，PD)非人灵长类(NHP)模型构建成功。
B.对帕金森病(Parkinson’s disease，PD)非人灵长类(NHP)进行DAP细胞移植由干细胞产生的DAP分化细胞在帕金森氏病(PD)的猴模型中进行评价。
1.在这项研究中，根据上文所描述的方法使猴干细胞分化以产生猴WT(猴iPSC细胞，猴血液PBMC通过CTSTM CytoTuneTM-iPS2.1仙台病毒重编程试剂盒(货号:A34546)制备得到)和猴DKO+G6分化的DAP细胞(猴DKO+G6-DAP细胞)。使用NHP-B2M-gRNA1和NHP-B2M-gRNA2对B2M外显子区段进行两端直接敲除，然后分别使用NHP-B2M-F/R对PCR引物进行基因组序列敲除验证。使用NHP-CIITA-gRNA1和NHP-CIITA-gRNA2对CIITA外显子区段进行两端直接敲除，然后分别使用NHP-CIITA-F/R对PCR引物进行基因组序列敲除验证,猴DKO+G6细胞过表达NHP-XSG006蛋白。
猴的DKO+G6细胞所用方法对应材料为：
NHP-B2M-gRNA1 CGTGAGTAAACCTGAATCTT
NHP-B2M-gRNA1 AGTCACATGGTTCACACGGC
NHP-CIITA-gRNA1 CATCGCTGTTGAGAAGCTCC
NHP-CIITA-gRNA1 GATATTGGCATAAGCCTCCC
B2M鉴定引物
NHP-B2M-F CATTTGGCCAGAGTGGAAATG
NHP-B2M-R TGGGACTCATTCAGGGTAGTA
CIITA鉴定引物
NHP-CIITA-F CTGTGAGGTGACTGAGCATATC
NHP-CIITA-R GGCCAGCAATGAGCATACTA
NHP-XSG006蛋白序列
2.确认低免疫原性iPS细胞衍生的DAP细胞标志物
免疫荧光检测显示猴WT及猴DKO+G6分化的DAP细胞在蛋白水平表达DAP特性基因：将细胞铺板在12孔板中，待细胞长到60-80％的密度后吸去培养基，加入4％多聚甲醛进行固定。细胞破膜后使用OTX2、EN1、LMX1A和FOXA2的一抗在4℃过夜孵育，洗去一抗后室温孵育带有荧光标记的二抗，随后使用荧光显微镜进行拍照。结果如图18所示，所有细胞均表达。
3.将低免疫原性猴DAP细胞注射到猴PD模型的脑部
将猴DAP细胞以立体定向方式两侧移植到MPTP处理的猴的壳核区，左侧注射猴WT-DAP细胞，右侧注射猴DKO+G6-DAP细胞。使用MRI的检测手段判定左右脑中DAP细胞的存活情况(图19)。MRI结果显示在28天的观察周期内，猴DKO+G6-DAP细胞在脑内持续存在，猴WT-DAP细胞在第五天即观察不到。
并根据本领域技术人员公认的方法来评价所移植的DA神经元的影响。用猴WT及猴DKO+G6分化的DAP细胞的猴中评价左右收行为、运动、PD症状、多巴胺能功能等的变化或差异(图20)。采食率结果显示，自造模成功后，双前肢细胞移植前完全不进行采食行为。移植细胞后，左前肢(猴DKO+G6-DAP注射影响侧)现已有好转迹象；右前肢(WT-DAP注射影响侧)无明显好转。
实施例5.正常非人灵长类(NHP)模型中的低免疫原性多巴胺能神经前体细胞
此实施例描述了一项在具有完全免疫系统的非人灵长类(NHP)模型上探究来自猴干细胞(iPSC)的体外分化的多巴胺能神经前体细胞(DAP)的免疫豁免能力的研究。对未造模的正常非人灵长类进行NHP-DAP细胞的同种异体移植，确认低免疫原性iPS细胞衍生的DAP可抵抗免疫系统攻击，使用MRI扫描和分析监测细胞的在体存活情况(参见例如Kikuchi等人，Nature，2017，548，592-596)。同批次猴WT-DAP细胞和猴DKO+G6-DAP细胞在免疫缺陷NOG小鼠中移植，确认DAP细胞体内安全性。并在PD-NOG小鼠中注射猴DKO+G6-DAP细胞，以确认低免疫原性DAP细胞体内分化特性。
A.对具有完全免疫系统的非人灵长类(NHP)进行同种异体DAP细胞移植，
将猴DAP细胞以立体定向方式两侧移植到壳核区，左侧注射猴WT-DAP细胞，右侧注射猴DKO+G6-DAP细胞。使用MRI的检测手段判定左右脑中DAP细胞的存活情况(图21A&B)。分别在移植前(pre-TX)、移植当天(day0)、移植后day7、14、21、28、42、56、90、122、152、183、212对猴脑进行MRI扫描。结果显示在259天的观察周期内，猴DKO+G6-DAP细胞在脑内持续存在，猴WT-DAP细胞在day14-21即观察不到。说明猴DKO+G6-DAP细胞具有低免性可逃逸免疫系统攻击，在具有完全免疫系统的同种异体猴脑内长期存活；而与此同时未经改造的WT-DAP细胞则在相同环境下被排斥无法存活。
B.对免疫缺陷的NOG小鼠进行NHP-DAP细胞移植
使用实施例3B的方法将猴WT-DAP细胞和猴DKO+G6-DAP细胞立体注射至不同NOG小鼠的左侧纹状体，移植后2个月对小鼠进行脑部取材，参照实施例3D的方法对NOG小鼠脑组织冰冻切片免疫荧光染色。对细胞移植3个月后小鼠的脑组织冠状冰冻切片进行hNA(红)&Ki67(绿)&DAPI(蓝)的共染(图22A)。染色结果显示，移植部位可见猴WT-DAP细胞和猴DKO+G6-DAP细胞，团块大小、数量在细胞间均无差异，两种细胞均只有极少量的Ki67占比(图22B)。体内安全性检测显示低免疫原性多巴胺能神经前体细胞(DKO+G6-DAP)与未编辑细胞(WT-DAP)相比具有相同正常的增殖能力，且均含有极少量的Ki67，体内致瘤性低。
C.对免疫缺陷的PD-NOG小鼠进行NHP-DAP细胞移植
使用实施例3A的方法对NOG小鼠进行PD造模，成模小鼠立体定位缓慢注射猴DKO+G6-DAP细胞进左纹状体。移植后3个月对小鼠进行脑部取材，参照实施例3D的方法对PD小鼠脑组织冰冻切片免疫荧光染色。对细胞移植3个月后小鼠的脑组织冠状冰冻切片进行hNA(红)&TH(绿)&DAPI(蓝)的共染(图23)。染色结果显示，低免疫原性DAP细胞可在体内分化生成TH+，即有功能的多巴胺能神经元。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (18)

  1. 一种从免疫豁免性诱导性多能干细胞分化的分离的低免疫原性多巴胺能神经细胞，其特征在于，所述神经细胞包含：
    1)HLA-I和HLA-II人类白细胞抗原的表达降低或不表达；
    2)表达由CD47和CD24的功能结构域构建的融合蛋白；
    所述细胞能逃逸T细胞攻击及NK细胞的杀伤。
  2. 根据权利要求1所述的分离的低免疫原性多巴胺能神经细胞，其特征在于，所述免疫豁免性诱导性多能干细胞中使用基因编辑工具靶向编码HLA-I的一种或多种转录调节因子的一个或多个基因，以及编码HLA-II的一种或多种转录调节因子的一个或多个基因，来实现HLA-I和HLA-II基因的表达降低或不表达；
    在某些实施方式中，为实现HLA-I和HLA-II基因的表达降低或不表达，所述HLA-I的转录调节因子可优选自：B2M、TAP1、TAP2、TAP相关糖蛋白(Tapasin)或NLRC5中的一个或多个；所述HLA-II的转录调节因子可优选自：CIITA、RFXANK、RFX5、RFXAP中的一个或多个；
    根据本发明，所述转录调节因子优选B2M和CIITA。
  3. 根据权利要求2所述的分离的低免疫原性多巴胺能神经细胞，其特征在于，所述免疫豁免性诱导性多能干细胞还包括通过选择性灭活CIITA基因的稀有切割核酸内切酶靶向CIITA基因的遗传修饰。
  4. 根据权利要求2所述的分离的低免疫原性多巴胺能神经细胞，其特征在于，所述免疫豁免性诱导性多能干细胞还包括通过选择性灭活B2M基因的稀有切割核酸内切酶靶向B2M基因的遗传修饰。
  5. 根据权利要求3或4所述的分离的低免疫原性多巴胺能神经细胞，其特征在于，其中通过稀有切割内切核酸酶靶向CIITA基因或B2M基因的遗传修饰包括CAS蛋白或编码CAS蛋白的多核苷酸，和至少一个用于特异性靶向CIITA基因或B2M基因的导向核糖核酸序列；
    在具体的实施方式中，使用CRISPR/CAS9系统分别对B2M和CIITA外显子区段进行两端直接敲除，其中，针对B2M基因的gRNA的靶序列为SEQ ID NO:2和SEQ ID NO:3，针对CIITA基因的gRNA的靶序列为SEQ ID NO:4和SEQ ID NO:5。
  6. 根据权利要求1或2所述的分离的低免疫原性多巴胺能神经细胞，其特征在于，所述免疫豁免性诱导性多能干细胞中通过引入针对编码HLA-I的一种或多种转录调节因子的一个或多个基因，或编码HLA-II的一种或多种转录调节因子的一个或多个基因的基因表达修饰分子，来实现HLA-I和/或HLA-II基因的表达降低或不表达，其中所述基因表达修饰分子包括选自siRNA，shRNA，microRNA，反义RNA和另一种RNA介导的抑制分子中的一种。
  7. 根据权利要求1所述的分离的低免疫原性多巴胺能神经细胞，其特征在于，所述包含CD47和CD24的功能结构域的融合蛋白中的CD47的功能结构域为CD47跨膜结构域；优选地，所述CD47跨膜结构域的氨基酸序列如SEQ ID NO：6-10任一项所示；
    所述包含CD47和CD24的功能结构域的融合蛋白中的CD24的功能结构域为CD24的信号肽序列、CD24成熟肽、CD24胞外肽、CD24膜锚定序列、CD24胞外成熟肽；优选地，所述CD24的功能结构域的氨基酸序列如SEQ ID NO：11-16任一项所示；
  8. 根据权利要求7所述的分离的低免疫原性多巴胺能神经细胞，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47 SIRPα结合域和CD24的膜锚定序列连接；或者，
    在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域插入至CD24的胞外序列和膜锚定序列的链接位点；或者，
    在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47链接到CD24的胞外序列后；或者，
    在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24成熟肽链接到CD47的SIRPα结合域后；或者，
    在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24的胞外成熟肽插入到CD47的SIRPα结合域和跨膜结构域链接点；或者
    在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24的成熟肽序列和CD47跨膜结构域连接；
    优选地，所述由CD47和CD24的功能结构域构建的融合蛋白的氨基酸序列与如SEQ ID NO:1所示的序列具有70％以上的同源性，例如80％以上的同源性，再例如90％以上、95％以上、98％以上的同源性；
    进一步优选地，所述由CD47和CD24的功能结构域构建的融合蛋白的氨基酸序列如SEQ ID NO:1所示；
    在具体的实施方式中，增加的CD47和CD24的功能结构域构建的融合蛋白表达是在所述的免疫豁免性诱导性多能干细胞中将至少一个拷贝的融合蛋白基因导入所述iPSC中处于启动子的控制下而产生的。
  9. 编码由CD47和CD24的功能结构域构建的融合蛋白的核酸序列可以根据权利要求8所述融合蛋白的氨基酸序列得到。
  10. 权利要求1-9任一项所述的分离的低免疫原性多巴胺能神经细胞选自多巴胺能神经前体细胞、多巴胺能神经祖细胞、未成熟的多巴胺能神经细胞和成熟的多巴胺能神经细胞；优选为多巴胺能神经前体细胞。
  11. 一种通过体外分化从免疫豁免性诱导性多能干细胞获得权利要求1-10任一项所述的分离的低免疫原性多巴胺能神经前体细胞的方法，其中在免疫豁免性iPSC细胞中已消除内源性β-2微球蛋白(B2M)基因活性和内源性II类反式激活因子(CIITA)基因活性；并且表达由CD47和CD24的功能结构域构建的融合蛋白；优选地，所述由CD47和CD24的功能结构域构建的融合蛋白的氨基酸序列与如SEQ ID NO:1所示的序列具有70％以上的同源性，例如80％以上的同源性，再例如90％以上、95％以上、98％以上的同源性；进一步优选地，所述由CD47和CD24的功能结构域构建的融合蛋白的氨基酸序列如SEQ ID NO:1所示，其特征在于，所述方法包括：
    免疫豁免性诱导性多能干细胞分化为低免疫原性多巴胺能神经前体细胞：
    (1)免疫豁免性诱导性多能干细胞的培养：
    按照约10万每孔的数量将免疫豁免性诱导性多能干细胞以团块的形式在预先包被过Laminin的基质上进行铺板，并使用E8完全培养基进行细胞培养，培养过程中每隔4～5天消化传代；
    (2)低免疫原性多巴胺能神经前体细胞的分化：
    S1：分化前一天，将汇合度约为70％～80％的免疫豁免性诱导性多能干细胞用TrypLE酶消化成单细胞，将细胞以8000/cm2的密度接种到预先包被过Vitronectin(Gibco)的6孔板中，接种培养基为StemFit完全培养基，并添加10μM Y27632组分以促进干细胞存活；
    S2：细胞贴壁生长24h后，将培养基更换为第一培养基，作为分化的第0天，称为D0，于37℃、5％CO2的二氧化碳细胞培养箱中培养8天，每两天换液一次；
    S3：在分化的第9天，用Accutase(Innovative)消化细胞，然后用第二培养基重悬细胞，再将1500万细胞接种至T75悬浮培养瓶中，摇晃均匀后，于37℃、5％CO2的二 氧化碳细胞培养箱中培养2天，使细胞在悬浮状态中自发形成神经球，得到中脑底板细胞；
    S4：在分化的第11天，将培养基更换为第三培养基，于37℃、5％CO2的二氧化碳细胞培养箱中培养5天，每2.5天换液一次，得到多巴胺能神经祖细胞；
    S5：在分化的第16天，将培养基更换为第四培养基，并将500万神经球细胞做贴壁处理，贴于Vitrovectin(Gibco)预先包被的T75培养瓶中继续培养，于37℃、5％CO2的二氧化碳细胞培养箱中培养7天，每2.5天换液一次；
    S6：在分化的第23天，用Accutase(Innovative)消化细胞，然后用第五培养基重悬细胞，再将1500万细胞接种至T75悬浮培养瓶中，摇晃均匀后，于37℃、5％CO2的二氧化碳细胞培养箱中培养一天，使细胞在悬浮状态中自发形成神经球，得到多巴胺能神经前体细胞(DAP细胞)；
    其中，第一培养基为含有1μM-20μM SB431542、1μM-3μM DMH1、0.2μM-1μM CHIR99021和1μM-1.75μM SAG的神经基础培养基；
    优选地，第一培养基为含有10μM SB431542、2μM DMH1、0.6μM CHIR99021和1μM SAG的神经基础培养基；
    第二培养基为含有5μM-15μM Y27632、1μM-20μM SB431542、1-3μM DMH1、0.2μM-1μM CHIR99021和1μM-1.75μM SAG的神经基础培养基；
    优选地，第二培养基为含有10μM Y27632、10μM SB431542、2μM DMH1、0.6μM CHIR99021和1μM SAG的神经基础培养基；
    第三培养基为含有0.8μM-1.2μM DMH1、0.1μM-0.5μM SAG和80ng/mL-120ng/mL成纤维细胞生长因子8b(FGF8b)的神经基础培养基；
    优选地，第三培养基为含有1μM DMH1、0.2μM SAG和100ng/mL成纤维细胞生长因子8b(FGF8b)的神经基础培养基；
    第四培养基为含有0.1μM-0.5μM SAG和80ng/mL-120ng/mL FGF8b的神经基础培养基；
    优选地，第四培养基为含有0.2μM SAG和100ng/mL FGF8b的神经基础培养基；
    第五培养基为含有5μM-15μM Y27632、0.1μM-0.5μM SAG和80ng/mL-120ng/mL FGF8b的神经基础培养基；
    优选地，第五培养基为含有10μM Y27632、0.2μM SAG和100ng/mL FGF8b的神经基础培养基；
    所述神经基础培养基选自IMDM(Iscove's Modified Dulbecco's Medium)培养基、Eagle's Basal Medium(BME)培养基、GMEM培养基、MEM培养基、DMEM培养基，Ham's F-12培养基、RPMI1640培养基、Neurobasal培养基中的至少一种；
    优选的，所述神经基础培养基由以下组分组成：98％(v/v)DMEM/F12(Gibco)、1％(v/v)非必须氨基酸(Non-essential amino acid，Gibco)和1％(v/v)N2添加剂(N2 supplement，Gibco)。
  12. 一种治疗患有神经退行性疾病或病况的患者的方法，所述方法包括施用包含治疗有效量的权利要求1-10任一项所述的分离的低免疫原性多巴胺能神经细胞或根据权利要求11所述的方法制备的分离的低免疫原性多巴胺能神经细胞的组合物。
  13. 根据权利要求12所述的方法，其中所述组合物还包含治疗有效的载体；
    优选地，所述分离的低免疫性多巴胺能神经细胞在可生物降解的支架上。
  14. 根据权利要求12所述的方法，其中所述施用包括移植或注射。
  15. 根据权利要求12-14任一项所述的方法，其中所述神经退行性疾病或病况选自帕金森氏病、亨廷顿氏病和多发性硬化症；优选为帕金森氏病。
  16. 一种组合物，其特征在于，所述组合物包含权利要求1-10任一项所述的分离的低免疫原性多巴胺能神经细胞或根据权利要求11所述的方法制备的分离的低免疫原性多巴胺能神经细胞。
  17. 根据权利要求16所述的组合物，其特征在于，所述组合物包含权利要求1-10任一项所述的分离的低免疫原性多巴胺能神经细胞和一种或多种治疗剂，所述治疗剂包含肽、细胞因子、小分子化合物、大分子、ADC、抗体、纳米粒子、生物类似物、mRNA、中药、蛋白、疫苗、检查点抑制剂、丝裂原、生长因子、小RNA、双链RNA(double stranded RNA；dsRNA)、单核血细胞、饲养细胞、饲养细胞组分或其置换因子、包含一种或多种所关注多核酸的载体、抗体等。
  18. 权利要求1-10任一项所述的分离的低免疫原性多巴胺能神经细胞或根据第权利要求11所述的方法制备的分离的低免疫原性多巴胺能神经细胞在制备用于神经退行性疾病或病况治疗的产品中的应用；
    在某些方面，所述应用，其中所述神经退行性疾病或病况选自帕金森氏病、亨廷顿氏病和多发性硬化症；优选为帕金森氏病。
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