A method for generating neural stem cells by gene editing of human iPSCs

Abstract

The present invention provides a method for generating neural stem cells by gene editing human iPSCs, neural stem cells obtained by this method, and methods for culturing or maintaining the neural stemness of such stem cells. A new type of NSCs, termed 2K-iNSCs, is obtained by knocking out the MLLT3-binding, ANK repeat, and PCGF1-interacting PUFD domain-encoding sequences (BCOR-MAP) of human iPSCs and the PLSCR1 gene. Compared to conventional methods, the induction and maintenance of 2K-iNSCs does not depend on NSC medium supplemented with small molecules or protein factors; instead, they can be passaged continuously in iPSC medium alone, maintaining their neural stemness. The present invention also provides sgRNAs and plasmids for knocking out the BCOR-MAP and PLSCR1 genes in iPSCs, and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to a method for producing neural stem cells by gene editing human iPSCs (induced pluripotent stem cells). [Background technology]

[0002] Neural stem cells (NSCs) possess the ability to self-renew and differentiate into various neuronal and glial cell types, making them valuable for in vitro disease modeling, drug screening, and regenerative medicine. Currently, NSCs can be obtained via three main methods: isolation from primary tissues, transdifferentiation from somatic cells, and differentiation from pluripotent stem cells. Obtaining NSCs from human pluripotent stem cells, particularly human iPSCs, offers advantages such as freedom from cell origin limitations and the ability to perform autologous transplantation. Traditional neural differentiation involves the aggregation or formation of embryoid bodies (EBs) from pluripotent stem cells (PSCs), but this process is prone to batch-to-batch variations (Tao and Zhang, 2016). The conventional solution is to add SB431542 and Noggin to suppress the SMAD-dependent TGFβ (Transforming growth factor beta) and BMP signaling pathways, further preventing the differentiation of extra embryonic and mesodermal tissues; this method achieves an induction efficiency of approximately 80% (Chambers et al., 2009). The most common method for inducing iPSCs into NSCs is to use NSC induction medium supplemented with small molecules, but this method has drawbacks, such as incomplete conversion of iPSCs into NSCs, high cellular heterogeneity among NSCs, and the risk of tumorigenesis.

[0003] Currently, NSCs derived from human iPSCs require the use of specific media to maintain their NSC characteristics during subculture. Unlike conventional NSC induction and maintenance methods, NSCs derived by knockout of the PLSCR1 gene and the MLLT3-binding, ANK repeat, and PCGF1-interacting PUFD domain sequence encoding BCOR (BCOR-MAP) in iPSCs can maintain their NSC characteristics simply by using iPSC media. These NSCs offer advantages such as high purity, low cellular heterogeneity, robust proliferation potential, long-term in vitro culture capability, and reduced spontaneous differentiation.

[0004] Methods for isolating and obtaining NSCs from human tissues have drawbacks, including limited sources, high heterogeneity of isolated NSCs, and limited quantity and expansion capacity. Methods for transdifferentiating and obtaining NSCs from somatic cells have drawbacks, including limited cell numbers and relatively low transdifferentiation efficiency due to the relatively weak proliferative capacity of somatic cells and limited number of generations. NSCs obtained using commercially available media supplemented with small molecular weight compounds or protein factors have drawbacks, including high heterogeneity and the risk of spontaneous differentiation and tumor formation. Furthermore, media for inducing and maintaining NSCs require the addition of various small molecular weight compounds or protein factors, limiting basic research and clinical applications of NSCs. Therefore, in conventional methods, medium components play an important role in the efficiency of inducing iPSCs into NSCs and maintaining their neural stemness. Summary of the Invention

[0005] The present invention provides a method for generating neural stem cells by gene editing human iPSCs. The present invention also provides neural stem cells obtained by this method and methods for culturing or maintaining their neural stemness. A new type of NSCs, termed 2K-iNSCs, is obtained by knocking out the MLLT3-binding, ANK repeat, and PCGF1-interacting PUFD domain (BCOR-MAP) coding sequence and the PLSCR1 gene in human iPSCs. Compared to conventional methods, the induction and maintenance of 2K-iNSCs does not depend on NSC medium supplemented with small molecules or protein factors; instead, they can be passaged continuously in iPSC medium alone, maintaining their neural stemness. The present invention also provides sgRNAs, plasmids, and uses thereof for knocking out the BCOR-MAP and PLSCR1 genes in iPSCs.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions: The present invention provides sgRNAs for knocking out the BCOR-MAP and PLSCR1 genes in iPSCs. The sequences of the sgRNAs are shown in SEQ ID NOs: 1 to 4. BCOR-MAP refers to the MLLT3-binding, ANK repeat, and PCGF1-interacting PUFD domains of BCOR.

[0007] The present invention provides a plasmid for knocking out the BCOR-MAP and PLSCR1 genes in iPSCs, which can be obtained by ligating the above-mentioned sgRNA to a base plasmid.

[0008] Preferably, the base plasmid is the PX330 plasmid.

[0009] The present invention provides use of the above-mentioned sgRNA or the above-mentioned plasmid in producing neural stem cells.

[0010] The present invention provides a method for generating neural stem cells by gene editing human iPSCs, which is obtained by knocking out the BCOR-MAP and PLSCR1 genes in human iPSCs.

[0011] Preferably, the knockout of the BCOR-MAP and PLSCR1 genes in the human iPSCs is carried out using the above-mentioned sgRNA or the above-mentioned plasmid.

[0012] The present invention provides a method for generating neural stem cells by gene editing human iPSCs. (1) ligating the sgRNA to a base plasmid; (2) introducing the constructed plasmid into human iPSCs and culturing them in hiPSC medium; (3) screening the cells obtained in step (3) using puromycin; (4) picking monoclones, identifying whether the BCOR-MAP and PLSCR1 genes have been knocked out, and screening cells in which the BCOR-MAP and PLSCR1 genes have been knocked out to obtain NSCs.

[0013] Preferably, step (4) further includes detecting whether cells in which the BCOR-MAP and PLSCR1 genes have been knocked out express NSC markers, and selecting cells that express NSC markers to obtain NSCs.

[0014] Preferably, the base plasmid in step (1) is a PX330 plasmid, the method of introduction in step (2) is electroporation, the human iPSCs in step (2) are human iPSCs in good culture conditions, and the hiPSCs culture medium in step (2) is mTesR1 medium.

[0015] The present invention provides neural stem cells, which are prepared by the above-mentioned method.

[0016] The present invention provides a method for culturing the above-mentioned neural stem cells or maintaining neural stemness, which comprises culturing the neural stem cells using a hiPSCs medium.

[0017] Preferably, the hiPSCs medium is mTesR1 medium. [Effects of the Invention]

[0018] The present invention provides a method for generating neural stem cells (NSCs) by gene editing of human iPSCs, and the neural stem cells (NSCs) obtained by this method. These NSCs can be cultured solely in hiPSC medium (e.g., mTesR1 medium) without relying on conventional NSC media, and can be passaged multiple times in hiPSC medium, maintaining their characteristics. These cells may be used in the future to construct neuronal disease models and treat nervous system disorders. [Brief explanation of the drawings]

[0019] [Figure 1] 1 is a flowchart of the present invention. [Figure 2] Schematic diagram of CRISPR-Cas9 cleavage sites in the BCOR and PLSCR1 genes. [Figure 3] This is the PCR identification result of PLSCR1 knockout. [Figure 4] Sequencing results of BCOR-MAP knockout. [Figure 5] Morphological characteristics of 2K-iNSCs and the neural spheres they formed. [Figure 6] 2K-iNSCs express neural stem cell marker genes. [Figure 7] 2K-iNSCs can differentiate into neurons, astrocytes, and oligodendrocytes. [Figure 8] These are the results of the teratoma experiment. DETAILED DESCRIPTION OF THE INVENTION

[0020] In order to more clearly illustrate the objectives, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific examples. While conventional methods for converting hiPSCs into NSCs involve inducing differentiation using a medium supplemented with specific small molecules, the method of the present invention simultaneously knocks out BCOR-MAP and PLSCR1 in hiPSCs. While conventional media for culturing NSCs require specially prepared media, NSCs obtained by the method of the present invention can be cultured directly in the hiPSC medium, eliminating the need to prepare a special medium for culture, thereby reducing the complexity of the culture process.

[0021] Example 1 1. Experimental materials: PX330 plasmid (Addgene, 42230); iPSCs (CELLAPY); mTesR1 medium (Stemcell, 85850); electroporator (Lonza, 2B, AAB-1001); Nucleofector Kit 2 for human stem cells (Lonza, VPH-5022); Matrigel (Corning, 354277).

[0022] Neuronal spontaneous differentiation medium (Shi et al., 2012): Neurobasal 24ml 24 ml of DMEM / F12 N2 (100x) 250 μl B27(50x) 500μl BDNF (20 μg / ml) 50 μl GDNF (20 μg / ml) 50 μl cAMP (1 mM) 50 μl Pen / Strep (100x) 500ul Oligodendrocyte induction medium: DMEM / F12 49ml N1(100×) 500ul Biotin (200 μg / ml) 25 ul FDGF-AA (20 μg / ml) 50 ul NT3 (20 μg / ml) 50 ul cAMP (1 mM) 50 ul Pen / Strep (100x) 500ul During the first week of induction, 10 ng / ml bFGF was added, and during the second to fourth weeks of induction, 10 ng / ml IGF1 was added.

[0023] 2. Experimental Method: The present invention provides a method for gene editing human iPSCs to generate neural stem cells, which comprises the following steps (Figure 1): (1) Construction of CRISPR-Cas9 plasmid for knocking out the PLSCR1 gene and BCOR-MAP: Using a commonly used sgRNA design website, design sgRNAs (Table 1) for knocking out the BCOR-MAP and PLSCR1 genes and ligate them into the PX330 plasmid.

[0024] JPEG2026504296000001.jpg35170

[0025] (2) Culture healthy human iPSCs and introduce the constructed PX330 plasmid into the cells by electroporation. There are several methods for knocking out the BCOR-MAP and PLSCR1 genes, and the same goal can be achieved using the following different methods. a) Using chemical transfection, the constructed BCOR-MAP and PLSCR1 gene knockout plasmids are introduced into iPSCs. b) sgRNA sequences for BCOR-MAP and PLSCR1 gene knockout are constructed into viral vectors, the viruses are packaged, and iPSCs are infected with the packaged viruses. c) CRISPR-Cas9, BCOR-MAP, and sgRNA of the PLSCR1 gene are transcribed into RNA, which is then introduced into cells by methods such as electroporation or chemical transfection. d) sgRNA RNA for the BCOR-MAP and PLSCR1 genes is synthesized and introduced into cells together with the Cas9 protein. e) Knock out the BCOR-MAP and PLSCR1 genes using ZFN (zinc finger nucleases), TALEN (TAL effector nucleases), etc.

[0026] (3) Screen the cells using puromycin.

[0027] (4) Monoclones are picked and subjected to PCR, followed by sequencing to identify whether the target gene has been knocked out, and NSC markers are detected by immunofluorescence.

[0028] (5) The obtained NSCs are subcultured, identified, and cryopreserved.

[0029] (6) The obtained NSCs are induced to differentiate to identify their differentiation potential.

[0030] (7) Teratoma experiments will be performed on the obtained NSCs to verify their transplant safety.

[0031] Test Results: (1) The obtained 2K-iNSCs possessed morphological characteristics of NSCs, were able to form neurospheres (Figure 5), and highly expressed NSC markers NESTIN, FABP7, and SOX2 (Figure 6). (2) 2K-iNSCs could be further differentiated into neurons, astrocytes, and oligodendrocytes (Figure 7). (3) When 2K-iNSCs were injected into the hindlimb muscle of NOD-SCID mice, no teratomas were formed (Figure 8). Hereinafter, the specific process and details of the present invention will be described in detail using "knockout of BCOR-MAP and PLSCR1 genes and induction of differentiation from iPSCs to 2K-iNSCs" as an example.

[0032] (1) Construction of BCOR-MAP and PLSCR1 gene knockout plasmids 1. Using the website https: / / www.benchling.com / crispr, we designed sgRNAs for knocking out the BCOR-MAP and PLSCR1 genes, selected sgRNA sequences with high scores, and sent them to a primer synthesis company for synthesis. 2. Plasmid PX330 (Addgene, 42230) purchased from Addgene was cleaved with the restriction enzyme BbsI, and the gel was collected. 3. The plus and minus strands of the synthesized sgRNA were annealed and then ligated to PX330 after restriction enzyme digestion.

[0033] (2) We knocked out and identified the BCOR-MAP and PLSCR1 genes in iPSCs. 1. iPSCs in good culture conditions (using mTesR1 medium) had a density of approximately 70-80%. 2. A 6-well plate was coated with Matrigel. 3. Remove the mTesR1 medium and electroporation reagents and preheat them. 4. The Matrigel in the coated 6-well plate was removed by aspiration, and 2 ml of mTesR1 containing 1 μM thiazolinone was added. The plate was then placed in a 37°C incubator to preheat. 5. Turn on the electroporator and adjust the program to use program B-016. 6. The PX330 plasmid was added to the electroporation solution and mixed homogenously for later use. 7. 700 μl of Accutase was added to the wells containing cultured human iPSCs, and the cells were digested at 37°C for approximately 5 minutes. After neutralization by adding 800 μl of DMEM / F12, the cells were dispersed by pipetting, aspirated into 1.5 ml of EP, and centrifuged at 200 g for 5 minutes. 9. The supernatant was aspirated, and the cells were resuspended in the plasmid electroporation solution mixture and then added to the electroporation cup. Care was taken to avoid introducing air bubbles. 10. Electroporation was performed. 11. After electroporation, the cells were resuspended in 1 ml of mTesR1 and added to a well of a Matrigel-coated 6-well plate. The medium was changed on day 12.2. 13. After the cells grew to a certain density, puromycin was added to screen the cells. 14. The medium was changed once a day. 15. The cells were cultured until they formed clones of appropriate size, which were then picked and cultured in 24-well plates. 16. Once the cells had proliferated to a certain number, a portion of the cells was harvested and the genome extracted. Primers were designed at both ends of the homology arms (Table 2) and PCR and sequencing were performed to identify the PLSCR1 knockout status. Homozygous knockout (2033 bp), heterozygous knockout (2033 bp, 663 bp), and wild-type (WT, 663 bp) were identified. The results indicated that the PLSCR1 gene in 2K-iNSCs was knocked out (Figure 3). Sequencing revealed that the BCOR gene of 2K-iNSCs contained a 7-bp sequence insertion that could potentially cause a frameshift mutation, indicating that the coding sequence for BCOR-MAP had already been knocked out (Figure 4). Immunofluorescence identification showed that 2K-iNSCs highly expressed NSC markers NESTIN, FABP7, and SOX2 (Figure 6).

[0034] JPEG2026504296000002.jpg38170

[0035] (3) In vitro identification of the differentiation potential of 2K-iNSCs 1. Spontaneous differentiation 1) Cell coverslips were placed in a 24-well plate and coated with Matrigel. 2K-iNSCs were seeded in the wells and cultured overnight in mTesR1 medium. 2) On day 2, the medium was replaced with neuronal spontaneous differentiation medium, and the medium was replaced every 2 days. 3) After 20 days of differentiation, the differentiated neurons were identified by immunofluorescence. The results showed that the differentiated 2K-iNSCs highly expressed the neuronal markers MAP2, NEUN, and TUJ1 (Figure 7A) and the glial cell marker GFAP (Figure 7B).

[0036] 2. Oligodendrocyte differentiation induction 1) Cell coverslips were placed in a 24-well plate and coated with Matrigel. 2K-iNSCs were seeded in the wells and cultured overnight in mTesR1 medium. 2) On day 2, the medium was changed to oligodendrocyte differentiation medium, and the medium was changed every 2 days. 3) During the first week of induction, 10 ng / ml bFGF was added, and during the second to fourth weeks of induction, 10 ng / ml IGF1 was added. 4) After the induction, the differentiated neurons were identified by immunofluorescence, and the results showed that the differentiated 2K-iNSCs highly expressed the oligodendrocyte marker O4 (Figure 7C).

[0037] (4) Verification of the safety of 2K-iNSC transplantation through teratoma experiments 1. The medium was removed by aspiration, and 500 μl of Accutase was added to each well (6-well plate), which was then placed in a 37°C incubator for 3 minutes to digest. 2. Accutase digestion was terminated by adding an equal volume of DMEM / F12, followed by centrifugation at 200 g for 5 minutes and removal of the supernatant. 3. The thawed Matrigel was mixed with an equal volume of chilled DMEM / F12, and 150 μl of resuspended cells was aspirated. The resuspended cells were injected into the hind leg muscles of NOD-SCID mice. The results showed that teratomas were formed in mice injected with iPSCs cells, but not in mice injected with 2K-iNSCs (Figure 8).

[0038] Compared with conventional techniques, the 2K-iNSCs obtained by the present invention have advantages such as excellent stability and the use of only the iPSC medium mTesR1 for induction and maintenance of neural stemness. These advantages are due to the fact that NSCs are obtained by knocking out BCOR-MAP and PLSCR1 in hiPSCs, rather than by inducing NSCs using conventional induction medium.

[0039] Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention, and do not limit the protection scope of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, it should be understood that those skilled in the art can modify or equivalently replace the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

[0040] References: Chambers, SM, Fasano, CA, Papapetrou, EP, Tomishima, M., Sadelain, M., and Studer, L. (2009). Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature biotechnology 27, 275-280. Shi, Y., Kirwan, P., and Livesey, F.J. (2012). Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat Protoc 7, 1836-1846. Tao, Y., and Zhang, S.C. (2016). Neural Subtype Specification from Human Pluripotent Stem Cells. Cell stem cell 19, 573-586.

Claims

1. An sgRNA for knocking out BCOR-MAP and PLSCR1 genes in iPSCs, wherein the sequence of the sgRNA is set forth in SEQ ID NO: 1 to SEQ ID NO:

4.

2. A plasmid for knocking out the BCOR-MAP and PLSCR1 genes of iPSCs, wherein the plasmid is obtained by ligating the sgRNA described in claim 1 to a base plasmid, and preferably the base plasmid is a PX330 plasmid.

3. Use of the sgRNA of claim 1 or the plasmid of claim 2 or 3 in the production of neural stem cells.

4. A method for producing neural stem cells by gene editing human iPSCs, characterized in that the NSCs are obtained by knocking out the BCOR-MAP and PLSCR1 genes in human iPSCs.

5. The method according to claim 5, wherein the knockout of the BCOR-MAP and PLSCR1 genes of the human iPSCs is carried out using the sgRNA of claim 1 or the plasmid of claim 2 or 3.

6. A method for producing neural stem cells by gene editing human iPSCs, comprising: (1) Linking the sgRNA of claim 1 to a base plasmid; (2) introducing the constructed plasmid into human iPSCs and culturing them in hiPSCs medium; (3) screening the cells obtained in step (3) using puromycin; (4) picking monoclones, identifying whether the BCOR-MAP and PLSCR1 genes have been knocked out, and screening the cells in which the BCOR-MAP and PLSCR1 genes have been knocked out to obtain NSCs; A method characterized by:

7. The method of claim 6, wherein the step (4) further comprises detecting whether the cells in which the BCOR-MAP and PLSCR1 genes have been knocked out express NSC markers, and selecting cells that express the NSC markers to obtain NSCs.

8. 7. The method of claim 6, wherein the base plasmid in step (1) is a PX330 plasmid, the method of introduction in step (2) is electroporation, the human iPSCs in step (2) are human iPSCs in good culture condition, and the hiPSCs culture medium in step (2) is mTesR1 medium.

9. A neural stem cell, characterized in that it is produced by the method according to any one of claims 4 to 8.

10. 10. A method for culturing neural stem cells or maintaining neural stemness according to claim 9, characterized in that the method comprises culturing the neural stem cells using a hiPSCs medium.

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