Preparation method and application of high-purity neural stem cells

Abstract

The invention particularly discloses a preparation method and application of high-purity neural stem cells. The method comprises the following steps: firstly, acquiring haploid neural stem cells, injecting the haploid neural stem cells into blastocysts, and culturing to form chimera embryos; separating nervous tissues from the embryos and dissociating the nervous tissues into mixed cell populations; finally, the target cells are efficiently enriched through a magnetic separation technology by utilizing the functionalized magnetic nanoparticles with the surfaces modified with the neural stem cell specific antibodies. According to the method, the genetic homogeneity advantage of a haploid technology, the in-vivo bionic development environment of chimera embryos and the label-free and high-precision purification capacity of magnetic separation are creatively fused, and the key technical bottlenecks of low purity, high heterogeneity and complex genetic background in traditional neural stem cell preparation are solved. The invention also provides the high-purity neural stem cell population prepared by the method, the special functionalized magnetic nanoparticles and application of the functionalized magnetic nanoparticles. The obtained cell population is high in purity and stable in function, and has important application value in the fields of disease modeling, drug screening, cell therapy and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical engineering and regenerative medicine technology, and specifically relates to stem cell culture, isolation and purification technology. More specifically, the present application relates to a method for preparing high-purity, single-genetic-background functional neural stem cells by combining haploid embryo technology, chimeric embryo culture technology and nano-magnetic separation technology, as well as products and applications obtained therefrom. BACKGROUND

[0002] Neural stem cells (NSCs) are a kind of multipotent progenitor cells with self-renewal ability and can differentiate into neurons, astrocytes and oligodendrocytes. They have immeasurable potential in the research and treatment of neural system development, repair and neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease). However, the wide application of neural stem cells has always been limited by two major challenges: one is how to obtain a high-purity and functionally uniform NSCs population; the other is how to obtain NSCs with clear and stable genetic background for precise gene function research and reliable disease modeling.

[0003] At present, the sources of neural stem cells mainly include: 1) direct isolation from embryonic or adult brain tissue; 2) directed induction and differentiation from pluripotent stem cells (including embryonic stem cells and induced pluripotent stem cells). The first method is limited by the difficulty in obtaining cells, the difficulty in obtaining materials and the many ethical constraints; the second method has become the mainstream, but its differentiation system is complex, and the final obtained cell population is often a heterogeneous mixture of neurons, glial cells and undifferentiated stem cells. To obtain relatively pure NSCs, conventional techniques mainly rely on flow cytometry sorting (FACS) or immunomagnetic bead sorting (MACS), which are usually directed against surface markers of NSCs (such as CD133, PSA-NCAM, etc.). However, FACS has a greater impact on cell activity, and the equipment is expensive; the magnetic beads used in traditional MACS are relatively large in size, which may affect the subsequent function of the cells, and the sorting purity often hovers around 80%-90%, which is difficult to meet the high requirements of single-cell genomics, high-throughput screening and other frontier research on cell uniformity.

[0004] On the other hand, in the genetic research model, haploid cells are directly and efficiently used for gene screening and functional research due to their only one set of chromosomes. Scientists have successfully cultivated mouse haploid embryonic stem cells and proved that they can participate in embryonic development to form chimeras. This provides a theoretical possibility for obtaining somatic cells (including neural stem cells) with a single genetic background by using haploid cells. However, how to combine the genetic advantages of haploid cells with the efficient purification technology of neural stem cells to obtain a large number of high-purity haploid or near-diploid neural stem cells in a targeted manner is still a technical gap that has not been solved. In addition, nanomaterials, especially magnetic nanoparticles, have great potential in the field of biological separation. Compared with commercial magnetic beads, superparamagnetic nanoparticles (such as Fe3O4) with smaller size and precisely modified surface interact more gently with cells and are easy to achieve high-precision targeting. However, how to design the surface chemistry to specifically and efficiently bind fragile neural stem cells and integrate into a complete preparation process is a new exploration direction in this field. Therefore, there is an urgent need in the art for an innovative integrated technical solution that can simultaneously guarantee the high purity and high quality of neural stem cell populations from the root (genetic background) and the preparation terminal (physical purification) to meet the needs of basic research and clinical translation. SUMMARY

[0005] In view of the deficiencies in the prior art, the primary purpose of the present application is to provide a preparation method of high-purity neural stem cells, which can efficiently and gently obtain a neural stem cell population with a clear genetic background and extremely high cell purity.

[0006] Another purpose of the present application is to provide a high-purity neural stem cell population prepared by the above method, which has specific purity and karyotype characteristics.

[0007] Still another purpose of the present application is to provide a functionalized magnetic nanoparticle specially used for the above method.

[0008] To achieve the above purposes, the present application adopts the following technical solutions: In a first aspect, the present application provides a preparation method of high-purity neural stem cells, comprising the following steps: S1. obtaining haploid neural stem cells; S2. injecting the haploid neural stem cells into a blastocyst to obtain a chimeric embryo; S3. separating and extracting neural tissue from the chimeric embryo to obtain a mixed cell population; S4. co-incubating the mixed cell population with functionalized magnetic nanoparticles modified with a neural stem cell-specific surface marker antibody on the surface to form a cell-nanoparticle complex; S5. applying an external magnetic field to the cell-nanoparticle complex to perform magnetic separation, and collecting the adsorbed target cells to obtain the high-purity neural stem cells.

[0009] Preferably, in step S1, the haploid neural stem cells are obtained by screening after flow cytometry directed induction differentiation from haploid embryonic stem cells, or are directly isolated and cultured from the inner cell mass of a haploid blastocyst.

[0010] Preferably, in step S2, the number of haploid neural stem cells injected is 10-20, and the blastocyst is a mouse or rat blastocyst stage embryo.

[0011] Preferably, in step S4, the neural stem cell specific surface marker is at least one of CD133, PSA-NCAM, or CD15.

[0012] Preferably, the functionalized magnetic nanoparticles are streptavidin-coated ferroferric oxide nanoparticles, and the antibody is a biotinylated monoclonal antibody.

[0013] More preferably, in step S4, the ratio of the functionalized magnetic nanoparticles to the mixed cell population is 0.5-2.0 µg: 1×10 5 cells, and the co-incubation conditions are 4-10°C, gentle shaking for 10-30 minutes.

[0014] Preferably, in step S5, the magnetic separation is performed using a separation column placed in a magnetic field, which includes washing the unbound impurity cells with a buffer, and eluting the target cells after removing the magnetic field.

[0015] In a second aspect, the present application provides a high-purity neural stem cell population obtained by the preparation method of the first aspect, wherein the proportion of cells expressing neural stem cell markers Nestin and Sox2 in the neural stem cell population is not less than 95%, and the haploid karyotype retention rate is not less than 80%.

[0016] In a third aspect, the present application provides a functionalized magnetic nanoparticle for use in the preparation method of the first aspect, which comprises a magnetic nanoparticle core, a silica layer coated on the core, and a biotin-streptavidin-biotinylated antibody complex structure connected to the silica layer, wherein the antibody is a monoclonal antibody targeting a neural stem cell specific surface marker.

[0017] In a fourth aspect, the present application provides a use of the high-purity neural stem cell population of the second aspect in constructing a nervous system disease model or performing high-throughput drug screening.

[0018] Compared with the prior art, the present application has the following remarkable advantages and beneficial effects: 1. Creative combination of source genetic advantage and terminal purification technology: The present application first systematically integrates "haploid chimera embryo technology" and "functionalized magnetic nanoparticle sorting technology". The former ensures the singleness and traceability of the genetic background from the cell source, and the latter provides a high-efficiency, label-free and low-damage physical purification method. The two work together to solve the fundamental contradiction between genetic background and cell purity that cannot be reconciled by traditional methods.

[0019] 2. Greatly improved purity and quality of prepared neural stem cells: Due to the use of in-vivo biomimetic chimeric embryo development environment, the neural stem cells have undergone a differentiation and maturation process closer to the physiological state, and their functional state is better. Combined with high-specificity nanomagnetic sorting, the proportion of Nestin / Sox2 double-positive cells in the final obtained cell population is stably above 95%, which is significantly higher than the traditional MACS (usually < 90%) or direct culture after differentiation (usually < 70%) method.

[0020] 3. Effective maintenance of haploid characteristics: The preparation system of the present application is gentle to cells, successfully maintaining the high proportion of haploid karyotype characteristics of the original haploid neural stem cells to the final product (retention rate ≥ 80%), which provides a unique tool for using such cells for efficient gene knockout, mutation screening and functional genomics research.

[0021] 4. Gentle and efficient sorting process, good cell activity: Fe3O4 nanoparticles with small size and good biocompatibility are used, and through low-temperature gentle incubation conditions, the damage to the cell membrane and activity during the sorting process is minimized. The survival rate of the recovered cells is higher than 95%, which is much better than flow cytometric sorting, and the operation is simple and fast, without the need for expensive equipment.

[0022] 5. Wide application prospect: The obtained high-purity, genetically clear neural stem cells are excellent seed cells for constructing precise nervous system disease models (such as introducing specific mutations in a haploid background using CRISPR), performing high-throughput neural activity drug or toxic compound screening, and developing high-quality cell therapy products, which have great scientific research and commercial value. DETAILED DESCRIPTION

[0023] The technical solutions of the present application are further described in detail through specific examples. It should be understood that the examples described herein are only for the purpose of explaining the present application, and not limiting the present application. All simple modifications, modifications or equivalent replacements based on the essence of the present application fall within the scope of protection of the present application.

[0024] The reagents and instruments used in the present application can be purchased through commercial channels unless otherwise specified. The biological experimental operations involved in the present application all comply with relevant ethical norms.

[0025] 1. Obtaining haploid neural stem cells: Take haploid embryonic stem cells (haESC) of C57BL / 6 mice cultured to the 3rd generation and in good condition, and perform suspension culture using a neural induction medium containing bis-nicotinamide (N2), B27, bFGF (20 ng / mL), and EGF (20 ng / mL) to form a pseudo-embryoid. After 7 days of culture, the pseudo-embryoid is attached and cultured until a large number of neural epithelial-like rosette structures appear. The cells are dissociated with Accutase enzyme, stained with a FITC-labeled anti-CD133 antibody, and sorted into a CD133-positive cell group by flow cytometry. The cell group is inoculated into a culture dish coated with polyornithine / laminin, expanded using a neural stem cell maintenance medium (N2 / B27 + bFGF + EGF), and haploid neural stem cells (haNSCs) are obtained. Karyotype analysis shows that the haploid proportion is 92%.

[0026] 2. Construction and culture of chimeric embryos: Collect Kunming white mouse blastocyst stage embryos (E3.5). Using a micromanipulator, about 15 haNSCs obtained in step 1 are injected into the blastocyst cavity of the blastocyst. The injected blastocyst is transferred to embryo culture solution and cultured in a 37°C, 5% CO2 incubator for 48 hours. Early primitive gut-like chimeras with obvious neural tube structures are observed.

[0027] 3. Isolation of neural tissue and dissociation of cells: Under a stereomicroscope, the chimeric embryo described above is finely dissected with a microneedle to isolate the developing neural tube and surrounding neural epithelial tissue. The tissue block is collected into a centrifuge tube and washed twice with pre-cooled PBS. Digestion solution containing 0.25% trypsin and 0.02% EDTA is added, and the solution is digested at 37°C for 5 minutes. Digestion is terminated by adding DMEM / F12 medium containing 10% fetal bovine serum, and the solution is gently blown into a single cell suspension and passed through a 40 μm cell strainer to obtain a mixed cell group.

[0028] 4. Preparation of functionalized magnetic nanoparticles: Fe3O4 magnetic nanoparticles with a diameter of about 50 nm were prepared by hydrothermal method. A layer of silica (SiO2) shell with a thickness of about 10 nm was coated on the surface of the nanoparticles by Stöber method. Subsequently, amino groups were introduced on the surface of the SiO2 by using silane coupling agent APTES, and then streptavidin (SA) was covalently coupled to the amino groups by using a cross-linking agent, obtaining SA-modified magnetic nanoparticles (SA-MNPs). The SA-MNPs were incubated with biotinylated anti-mouse PSA-NCAM monoclonal antibody (Biotin-anti-PSA-NCAM) at a mass ratio of 1:0.2 at 4°C for 1 hour, and functionalized magnetic nanoparticles (Ab-MNPs) were prepared by high-affinity binding of biotin-streptavidin. The magnetic nanoparticles were separated by a magnetic stand, washed with PBS three times to remove free antibodies, and resuspended in PBS containing 0.1% BSA for use.

[0029] 5. Magnetic sorting and obtaining of high-purity neural stem cells: about 1 x 10 6 cells obtained in step 3 were centrifuged and resuspended in 100 μL of ice-cold sorting buffer (PBS + 0.5% BSA + 2 mM EDTA). Ab-MNPs suspension with an iron content of 10 μg was added. The mixed system was placed on a 4°C rotary shaker and incubated with gentle shaking for 20 minutes. The incubated mixture was added to an MS sorting column placed in a strong magnetic stand. After the liquid naturally flowed out, the sorting column was washed with 500 μL of sorting buffer three times to remove impurity cells not bound to the nanoparticles. The sorting column was removed from the magnetic stand and placed on a sterile collection tube, 1 mL of preheated complete medium was added, and the liquid in the column was quickly pushed out with a plunger. This eluate was a suspension rich in target neural stem cells. After centrifugation, the cells were resuspended in a culture dish for culture.

[0030] 6. Cell characterization: the sorted cells were identified as follows: Immunofluorescence staining: after fixation of the cell slides, double staining was performed with anti-Nestin and anti-Sox2 antibodies, and DAPI was used for nuclear staining. At least 500 cells were counted randomly, and the results showed that the proportion of Nestin and Sox2 double-positive cells was 96.7%. Flow cytometry analysis: the sorted cells were stained with PE-labeled anti-CD133 antibody, and flow cytometry detection showed that the proportion of CD125 positive cells was 95.2%.

[0031] Karyotype analysis: part of the cells were taken for chromosome smears and Giemsa staining, and the statistics showed that 85% of the cells still maintained haploid karyotype (n=20).

[0032] Functional verification: cells were seeded in differentiation medium (bFGF and EGF were removed and BDNF and GDNF were added) for 7 days, and successfully differentiated into Tuj1-positive neurons and GFAP-positive astrocytes, proving their multi-directional differentiation potential.

[0033] This example aims to verify the effectiveness of directly isolating neural stem cells from haploid blastula, and compare it with the induced differentiation method of Example 1.

[0034] Group A: the same haploid ESC induced differentiation method as Example 1 was used.

[0035] Group B: mouse haploid blastula (developed by combining sperm with enucleated oocytes) was obtained. The haploid blastula was seeded on a mitomycin C-treated mouse embryonic fibroblast feeder layer, cultured using the haESC culture system, and haploid inner cell mass clones were obtained. After picking the clones and digesting them into single cells, they were directly cultured and expanded using neural stem cell conditioned medium (N2 / B27 + bFGF + EGF). Morphology (formation of typical neural spheres) and immunostaining (Nestin-positive) were used to identify haploid neural stem cells.

[0036] The haNSCs obtained from groups A and B were prepared according to the same procedures as steps 2 to 5 in Example 1 (the same number was injected into wild-type blastula, and the same anti-PSA-NCAM Ab-MNPs were used for sorting).

[0037] Results: The Nestin / Sox2 double-positive rate of the final product of group A was 96.7%, and the haploid karyotype retention rate was 85%; the Nestin / Sox2 double-positive rate of the final product of group B was 94.8%, and the haploid karyotype retention rate was 88%. There was no statistically significant difference in purity and function between the two groups (p>0.05), indicating that both sources of haploid NSCs were feasible. However, the operation of group B was more direct and the cycle was slightly shorter.

[0038] This example explores the effect of antibody-modified magnetic nanoparticles targeting different surface markers on sorting efficiency. Three different functionalized magnetic nanoparticles were prepared: biotinylated anti-CD133, anti-PSA-NCAM, and anti-CD15 antibodies were coupled respectively. The same batch of chimeric embryo mixed cell population prepared by the method of Example 1 was used as the sorting object.

[0039] The sorting process was the same as step 5 of Example 1. Three sorting products were respectively: 1. Flow cytometry was used to detect the positive rates of CD133, PSA-NCAM, and CD15.

[0040] 2. Immunofluorescence staining was used to count the Nestin / Sox2 double-positive rate.

[0041] 3. CCK-8 method was used to detect cell viability 24 hours after sorting.

[0042] Results: Anti-CD133 Ab-MNPs sorting group: CD133 positive rate was 98.1%, Nestin / Sox2 double positive rate was 95.9%, and cell viability was 96.5%.

[0043] Anti-PSA-NCAM Ab-MNPs sorting group: PSA-NCAM positive rate was 97.5%, Nestin / Sox2 double positive rate was 96.7%, and cell viability was 97.2%.

[0044] Anti-CD15 Ab-MNPs sorting group: CD15 positive rate was 90.3%, Nestin / Sox2 double positive rate was 88.4%, and cell viability was 95.8%.

[0045] Conclusion: The Ab-MNPs prepared using anti-CD133 or anti-PSA-NCAM antibodies had better sorting purity and cell viability than the anti-CD15 antibody group. The anti-PSA-NCAM group was slightly better in terms of purity and viability, and can be used as the preferred marker.

[0046] This example investigates the effect of the size of the magnetic nanoparticle core and the surface modification method on the sorting performance.

[0047] Four different functionalized particles were prepared: C1: The core was 20 nm Fe3O4, and after SiO2 coating, the anti-PSA-NCAM antibody was directly covalently coupled (without SA-Biotin system).

[0048] C2: The core was 50 nm Fe3O4, and after SiO2 coating, the anti-PSA-NCAM antibody was directly covalently coupled.

[0049] C3: The core was 50 nm Fe3O4, and the anti-PSA-NCAM antibody was connected using the SA-Biotin system of Example 1.

[0050] C4: The core was 100 nm Fe3O4, and the anti-PSA-NCAM antibody was connected using the SA-Biotin system of Example 1.

[0051] The same batch of mixed cells was used, and the total amount of iron element in the particles was fixed at 10 µg / 1×10 6Sort the cells and perform a sorting experiment. Evaluation indicators: 1) sorting purity (Nestin+ %); 2) cell recovery rate (number of live cells obtained by sorting / estimated number of NSCs in the theoretically mixed cell population x 100%); 3) cell apoptosis rate after sorting (Annexin V-PI double staining method).

[0052] Results: Conclusion: The particle with a core of 50 nm reaches the best balance in magnetic responsiveness and cell interaction. The sorting purity and recovery rate of the antibody connected by the SA-Biotin system are significantly better than those of the direct coupling method, and the apoptosis stress caused to the cells is minimal. Therefore, the structure (50 nm Fe3O4@SiO2-SA-Biotin-Ab) described in Example 1 is the most preferred solution.

[0053] This example demonstrates the application of haploid neural stem cells obtained by the method of the present application to gene function screening.

[0054] 1. Construct a gene mutation library: use a lentivirus vector library to randomly introduce a CRISPR-Cas9 system carrying different sgRNAs into high-purity haploid neural stem cells prepared and expanded by the method of Example 1, to construct a gene knockout mutation cell pool.

[0055] 2. Apply screening pressure: divide the mutation cell pool into two groups, one group is cultured in normal medium (control group), and the other group is cultured in medium containing a specific neurotoxic compound (such as MPP+, a Parkinson's disease model toxin) (screening group), and cultured for 5 days.

[0056] 3. Screen drug-resistant / sensitive genes: collect the surviving cells of the two groups, extract genomic DNA, amplify the sgRNA sequence by PCR and perform high-throughput sequencing. Compare the abundance changes of each sgRNA sequence in the two groups. The sgRNA significantly enriched in the screening group corresponds to the "neuroprotective gene" that can confer the ability of the cell to resist the toxin after knockout; and the significantly reduced one is considered to be an "essential gene" necessary for cell survival.

[0057] 4. Verification: select an sgRNA with the highest enrichment degree screened out, reconvert haploid NSCs to obtain a cell line with homozygous knockout of the gene. Compared with wild-type cells, the knockout cell line has a significantly improved survival rate under MPP+ treatment, confirming the effectiveness of the screening results.

[0058] This example demonstrates that the haploid neural stem cells prepared by the present application, which have high purity and a single genetic background, are an ideal platform for efficient and reliable forward genetics screening of gene function.

[0059] Take the same strain of mice as in Example 1 diploid embryonic stem cells, using the exact same neural induction differentiation scheme (embryoid body method) to obtain neural stem cells. After differentiation, use commercial anti-PSA-NCAM microbeads and MACS sorting column, strictly according to the manufacturer's instructions for positive sorting. The sorted cells are characterized in the same way as in Example 1.

[0060] Results: Immunofluorescence showed that the proportion of Nestin / Sox2 double positive cells was 86.4%. The CD133 positive rate was 84.7% by flow cytometry. The cells were all normal diploid karyotype. The differentiation ability test showed no significant difference from Example 1 group.

[0061] Steps 1 to 3 of Example 1 were performed to obtain a mixed cell population from the chimera embryo. Instead of magnetic sorting, the mixed cell population was directly inoculated into neural stem cell maintenance medium, and most non-neural cells were removed by differential adhesion (30 minutes), followed by continuous culture and expansion for 3 generations to obtain relatively pure neural stem cells. The same characterization was performed.

[0062] Results: After multiple generations of culture and purification, the proportion of Nestin / Sox2 double positive cells increased to 81.5%. There were about 15% haploid cells in the cell population. This method is long in cycle, limited in purity improvement, and cannot effectively enrich haploid cells.

[0063] Comprehensive effect comparison: The core indicators of the final products of Example 1, Comparative Example 1 and Comparative Example 2 are as follows: Conclusion: Through the innovative integration of the present application, the cell purity and genetic property maintenance have been significantly improved compared to existing single technologies, fully demonstrating the creativity, novelty and practical value of the present application.

Claims

1. A method for preparing high purity neural stem cells, characterized by, The method comprises the following steps: S1. obtaining haploid neural stem cells; S2. injecting the haploid neural stem cells into a blastocyst to obtain a chimera embryo; S3. isolating neural tissue from the chimera embryo and dissociating to obtain a mixed cell population; S4. co-incubating the mixed cell population with functionalized magnetic nanoparticles modified with antibodies specific to neural stem cell surface markers to form a cell-nanoparticle complex; S5. applying an external magnetic field to magnetically separate the cell-nanoparticle complex and collect the adsorbed target cells, thereby obtaining high-purity neural stem cells.

2. The production method according to claim 1, characterized by, In step S1, the haploid neural stem cells are obtained by screening after directed differentiation of haploid embryonic stem cells by flow cytometry, or are directly isolated and cultured from the inner cell mass of a haploid blastocyst.

3. The preparation method according to claim 1, characterized in that, In step S2, the number of injected haploid neural stem cells is 10-20, and the blastocyst is a mouse or rat blastocyst stage embryo.

4. The method of claim 1, wherein, In step S4, the neural stem cell specific surface marker is at least one of CD133, PSA-NCAM, or CD15.

5. The preparation method according to claim 4, characterized in that, The functionalized magnetic nanoparticles are streptavidin-coated ferroferric oxide nanoparticles, and the antibody is a biotinylated monoclonal antibody.

6. The production method according to claim 1 or 5, characterized by, In step S4, the functionalized magnetic nanoparticles are added to the mixed cell population in a ratio of 0.5-2.0 pg of iron element of the nanoparticles: 1 x 10 5 cells, and the incubation is performed at 4-10 °C with gentle shaking for 10-30 minutes.

7. The preparation method according to claim 1, characterized in that, In step S5, the magnetic separation is performed using a separation column placed in a magnetic field, which includes washing the unbound impurity cells with a buffer solution, and eluting the target cells after removing the magnetic field.

8. A population of high purity neural stem cells obtained by the method of any one of claims 1 to 7, wherein the population of neural stem cells comprises at least 90% neural stem cells. The proportion of cells expressing neural stem cell markers Nestin and Sox2 in the neural stem cell population is not less than 95%, and the haploid karyotype retention rate is not less than 80%.

9. Functionalized magnetic nanoparticles for use in the method of any one of claims 1 to 7, characterized in that, The functionalized magnetic nanoparticles are composed of a magnetic nanoparticle core, a silica layer coated outside the core, and a biotin-streptavidin-biotinylated antibody complex structure connected to the silica layer, wherein the antibody is a monoclonal antibody targeting neural stem cell specific surface markers.

10. Use of the high-purity neural stem cell population of claim 8 in constructing a nervous system disease model or performing high-throughput drug screening.