Stem cell treatment safety evaluation method based on brain organs

Abstract

The invention provides a brain organ-based stem cell treatment safety evaluation method, which comprises the following steps: S1, constructing a brain organ; s2, preparing cells to be detected, and marking fluorescent protein; s3, injecting the to-be-detected cells with the fluorescent protein markers into the brain organs; and S4, analyzing the to-be-detected cells injected into the brain organs. The invention finds that the dopaminergic cells can maintain normal growth and differentiation in brain organs; the stem cells can maintain high proliferation and dryness in brain organs; residual stem cells can be sensitively detected from brain organs, and the residual stem cells show higher proliferation and dryness in brain tumor organs; meanwhile, compared with mouse brain and whole brain organs, early-stage dopamine precursor cells also show higher proliferation ability in brain tumor organs. In conclusion, the brain organ can be used as a novel tool for safety quality inspection of cell therapy, and potential tumorigenic cells can be more sensitively detected from the brain tumor organ.

Description

Technical Field

[0001] This invention relates to a method for evaluating the safety of stem cell therapy, and more specifically, to a method for evaluating the safety of stem cell therapy based on brain organoids. Background Technology

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease, with a prevalence rate of 1.37% among Chinese people over 60 years of age, severely increasing the burden on families and society. Currently, the main clinical treatments are drug therapy centered on levodopa and deep brain stimulation (DBS). However, these treatments only improve PD symptoms and cannot stop the progressive death of dopaminergic neurons. Therefore, research into new treatment methods is urgently needed. In recent years, with the continuous development of stem cell technology, cell therapy has become a promising new approach for PD treatment. Human pluripotent stem cells (hPSCs) offer unprecedented opportunities for cell therapy due to their unlimited self-renewal and pluripotent differentiation capabilities. However, residual hPSCs in cell products and undifferentiated neural progenitor cells with strong proliferative capacity can lead to tumor formation, and their potential tumorigenicity is a significant obstacle to the clinical translation of stem cell therapy. Therefore, safety testing of stem cell therapy is the "first line of defense" before entering clinical trials and is a primary indicator in clinical trials.

[0003] Traditional tumorigenicity testing is mainly based on tumorigenicity experiments in immunodeficient rodents. However, traditional animal tumorigenicity experiments have limitations, namely: (1) large species differences, animal experiments cannot fully predict human transplantation results. For example, tumors have been reported in patients with ataxia-telangiectasia who received neural stem cell transplantation, patients with lupus nephritis who received autologous hematopoietic stem cell transplantation, and diabetic patients who received pancreatic β-cell transplantation; (2) long animal experiment cycle. A complete mouse tumorigenicity experiment takes 9-12 months, resulting in a lengthy stem cell therapy process and patients not receiving timely and effective treatment; (3) large number of experimental animals, making it difficult to ensure animal welfare. In the preclinical safety experiments of PD cell therapy, the tumorigenicity experiment alone requires as many as 150-200 animals, increasing treatment costs and making it difficult to ensure the welfare of experimental animals. Based on the above limitations of animal quality inspection, we intend to construct a new safety evaluation tool that more realistically reflects the human brain.

[0004] In 2013, Lancaster et al. successfully cultured hPSCs under suspension conditions to produce three-dimensional cultures with brain-like structural features, which they named brain organoids. Brain organoids have cell composition and anatomical structures highly similar to the human brain, and can reproduce the physiological, pathological, and pharmacological characteristics of the human brain, making them a new tool for exploring neurodevelopment, mental illnesses, neurodegenerative diseases, and drug screening.

[0005] The content of the background section is merely the technology known to the inventor and does not necessarily represent the prior art in this field. Summary of the Invention

[0006] The purpose of this invention is to establish a new method for safety evaluation of stem cell therapy based on brain organoids, and to explore which brain organoids can better detect cell products with potential tumorigenic risk.

[0007] To achieve the above objectives, the present invention provides a method for evaluating the safety of stem cell therapy based on brain organoids, comprising the following steps:

[0008] S1. Constructing brain organoids;

[0009] S2. Prepare the cells to be tested and label them with fluorescent proteins;

[0010] S3. Inject the test cells labeled with fluorescent protein into brain organoids;

[0011] S4. Analyze the test cells injected into brain organoids.

[0012] In some embodiments of the present invention, the brain organoid in S1 is a whole-brain organoid.

[0013] In some embodiments of the present invention, the brain organoid in S1 is a brain tumor organoid.

[0014] In some embodiments of the present invention, the brain tumor organoids are derived from TP53. - / - / PTEN - / - hPSCs.

[0015] In some embodiments of the present invention, the TP53 - / - / PTEN - / - hPSCs are obtained by knocking out the TP53 and PTEN genes in hPSCs using CRISPR / Cas9 technology.

[0016] In some embodiments of the present invention, the cells to be tested in S2 include one or more of the following: dopaminergic cells, stem cells, dopaminergic cells mixed with 1%-10% stem cells, and early dopaminergic precursor cells.

[0017] In some embodiments of the present invention, the fluorescent protein in S2 includes one or more of RFP, OFP, YFP, GFP, CFP, BFP, Luciferase, and tdTomato signal.

[0018] In some embodiments of the present invention, the fluorescent protein labeling method in step S2 is to add lentiviruses carrying RFP, OFP, YFP, GFP, CFP, BFP, Luciferase, and tdTomato signals to the cells to be tested and then culture them.

[0019] In some embodiments of the present invention, step S3 includes: routinely digesting and collecting the test cells labeled with fluorescent protein; aspirating 3-10 μL of cell suspension using a microinjection needle; aspirating the brain organoid from the dish and placing it in a culture dish, adding culture medium; injecting the test cells at a rate of 0.5-2 μL / min; after injection, returning the brain organoid to the culture dish, replacing the culture medium, and returning it to the carbon dioxide incubator.

[0020] In some embodiments of the present invention, the analytical method for the test cells injected into the brain organoid in step S4 includes immunofluorescence analysis and histological analysis.

[0021] The immunofluorescence assay marker is preferably one or more of the following: stem cell marker OCT4, dopaminergic cell marker FOXA2, TH, and proliferation marker Ki67.

[0022] The preferred histological analysis is H&E staining and / or immunohistochemistry.

[0023] Furthermore, the stem cells are hPSCs.

[0024] This invention discovers that dopaminergic cells grow and differentiate in brain organoids, while stem cells maintain high proliferative capacity and stemness. Furthermore, brain organoids exhibit high sensitivity for detecting residual stem cells; even a 1% presence of stem cells among dopaminergic cells can be detected. Residual stem cells show even higher proliferative capacity and stemness in brain tumor organoids, meaning brain tumor organoids are more sensitive to detecting residual stem cells. Simultaneously, brain tumor organoids also demonstrate higher proliferative capacity for detecting early dopamine precursor cells. This invention, through the brain organoid platform, provides a novel tool and method for safety testing in the field of cell therapy.

[0025] Compared to traditional mouse brain models, brain organoids have the following advantages: (1) High sensitivity: All experimental groups of brain organoids can detect 1% pluripotent stem cells mixed in the cell products and can maintain a high proliferation rate, while only 15%-33% of groups mixed with 1% stem cells in traditional animals can form tumors; (2) Simplicity: Brain organoid cell injection is simpler and organoid samples are easier to process; (3) Shorter observation time: Traditional animal experiments require at least six months to one year, but the enhanced ability of brain organoids to detect residual stem cells can shorten the experimental time; (4) Improved animal welfare. The supplementation of brain organoids can reduce unnecessary animal harm. Attached Figure Description

[0026] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0027] Figure 1 This is a flowchart of the brain organoid cell injection process described in this invention, where "Fluorescent protein label" represents fluorescent protein labeling; "Organoid injection" represents brain organoid injection; "hPSCs" represents human pluripotent stem cells; "immature mDA cells" represents early dopaminergic precursor cells; "mDA cells" represents dopaminergic cells; and "spiked hPSCs" represents dopaminergic cells from hybrid stem cells.

[0028] Figure 2 The present invention describes how dopaminergic cells can maintain normal growth and differentiation in brain organoids; dopaminergic cells injected into brain organoids are labeled as RFP (A); bright-field and in vivo imaging images of dopaminergic cells in whole-brain organoids (B) and brain tumor organoids (C) at 2 and 4 weeks after injection; scale bar: 1000 μm for low magnification and 200 μm for high magnification; D. Immunofluorescence images of FOXA2, TH, and Ki67 after injection into brain organoids; scale bar: 1000 μm for high magnification and 80 μm for low magnification; E. Immunofluorescence images of hNuMA, FOXA2, TH, Ki67, and SOX1 after transplantation of dopaminergic cells into NOD SCID mice; scale bar: 80 μm; F. Statistical graph of immunofluorescence markers among whole-brain organoids, brain tumor organoids, and mice;

[0029] Figure 3The present invention describes stem cells that maintain high proliferative capacity and stemness in brain organoids; RFP-labeled stem cells to be injected (A); bright-field and in vivo imaging images of stem cells injected into whole-brain organoids (B) and brain tumor organoids (C) 2 and 4 weeks later; low-power scale bar: 1000 μm, high-power scale bar: 200 μm; D. Immunofluorescence images of OCT4, Nestin, and Ki67 after stem cells are injected into brain organoids; high-power scale bar: 1000 μm, low-power scale bar: 80 μm; E. Immunofluorescence images of hNuMA, OCT4, Nestin, and Ki67 after stem cells are transplanted into mice; scale bar: 80 μm; F. Quantitative statistical graph of immunofluorescence markers among whole-brain organoids, brain tumor organoids, and mice;

[0030] Figure 4 This is a histological analysis of stem cells injected into brain organoids and mice as described in this invention; A. H&E staining images after stem cells are injected into brain organoids and mice; high magnification scale bar: 1000 micrometers, low magnification scale bar: 100 micrometers; B. Immunohistochemical images (showing RFP) after stem cells are injected into brain organoids; scale bar: 100 micrometers;

[0031] Figure 5 Image of Luciferase in brain organoids containing dopaminergic cells mixed with residual stem cells, as described in this invention; dopaminergic cells are labeled RFP, while stem cells are labeled Luciferase; a dopaminergic cell suspension mixed with 10% or 1% stem cells was prepared and injected into brain organoids (A); Luciferase signal was dynamically observed at 2, 4, 8, and 12 weeks after injection (B); C. Statistical graph of relative Luciferase expression among different groups;

[0032] Figure 6 The images show immunofluorescence patterns of dopaminergic cells mixed with residual stem cells injected into brain organoids according to the present invention; A. Immunofluorescence patterns of OCT4, FOXA2, and Ki67 after dopaminergic cells mixed with residual stem cells were injected into brain organoids; scale bar: 1000 μm for high magnification and 80 μm for low magnification; B. Immunofluorescence patterns of hNuMA, FOXA2, TH, Ki67, and SOX1 after dopaminergic cells mixed with residual stem cells were transplanted into mice; scale bar: 80 μm; C. Quantitative statistical graph of immunofluorescence markers among whole-brain organoids, brain tumor organoids, and mice;

[0033] Figure 7This invention relates to the histological analysis of dopaminergic cells mixed with residual stem cells injected into brain organoids; A. H&E staining images of dopaminergic cells mixed with residual stem cells injected into brain organoids and mice; high magnification scale bar: 1000 micrometers, low magnification scale bar: 100 micrometers; B. Immunohistochemical staining images of dopaminergic cells mixed with residual stem cells injected into brain organoids; scale bar: 100 micrometers;

[0034] Figure 8 The first image shows the high proliferation and growth of early dopaminergic precursor cells in brain tumor organoids as described in this invention; bright-field and in vivo imaging images at 2 and 4 weeks after injection of dopaminergic cells on day 15 of differentiation into whole-brain organoids (A) and brain tumor organoids (B); scale bar: 1000 μm for low-power scale and 200 μm for high-power scale; C. Immunofluorescence images of FOXA2, TH, and Ki67 after injection of early dopaminergic precursor cells into brain organoids; scale bar: 1000 μm for high-power scale and 80 μm for low-power scale; D. Immunofluorescence imaging images of hNuMA, FOXA2, TH, Ki67, and SOX1 after transplantation of early dopaminergic precursor cells into NOD SCID mice; scale bar: 80 μm; E. Quantitative statistical graph of immunofluorescence markers among whole-brain organoids, brain tumor organoids, and mice. Detailed Implementation

[0035] In the following description, only certain exemplary embodiments are depicted simply. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0036] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0037] Unless otherwise defined herein, scientific and technical terms used in conjunction with this invention will have the meaning commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context otherwise clearly indicates, the singular forms “a,” “an,” and “the” include plural indicators. In this application, unless otherwise stated, the use of “or” means “and / or.” Furthermore, the use of the term “comprising” and other forms such as “including” and “containing” is not limiting. Moreover, the scope provided in the specification and the appended claims includes all values ​​between endpoints.

[0038] definition

[0039] To better understand this invention, the definitions and explanations of relevant terms are provided below.

[0040] The term "organoid" refers to a miniature organ produced by three-dimensional (3D) culture of stem cells in vitro. It is an organ-like organism with the ability to self-renew and assemble, and has a structure and function that are highly similar to organs.

[0041] The term "human pluripotent stem cells (hPSCs)" refers to a type of cell that has the ability to self-renew and differentiate into various cell phenotypes in the human body.

[0042] The term "whole-brain organoids" refers to miniature organs with a three-dimensional structure that are highly similar to the real brain, cultivated in vitro using stem cell technology. These organoids can mimic the brain's developmental process and, to a certain extent, reproduce the brain's functions and characteristics.

[0043] The term "TP53 gene," also known as P53, is a tumor suppressor gene that encodes a protein with a molecular weight of 53 kDa. Mutations in this gene are found in more than 50% of all malignant tumors.

[0044] The term "PTEN gene" is the first tumor suppressor gene discovered to date with dual phosphatase activity. It has dual specificity as both a lipid phosphatase and a protein phosphatase and plays an important role in the regulation of multiple intracellular signal transduction pathways.

[0045] The term "TP53" - / - / PTEN - / - "Brain tumor organoids derived from hPSCs" are created by knocking out the TP53 and PTEN genes in human pluripotent stem cells (hPSCs) and then differentiating them into brain tumor organoids.

[0046] The term "brain organoids" is a general term for organoids involving the brain, including whole-brain organoids and brain tumor organoids.

[0047] The term "H&E staining" refers to hematoxylin-eosin staining, or HE staining for short, a commonly used staining method in paraffin sectioning. Hematoxylin is an alkaline staining solution, primarily causing the chromatin in the cell nucleus and nucleic acids in the cytoplasm to turn purple-blue; eosin is an acidic dye, primarily causing the components in the cytoplasm and extracellular matrix to turn red. HE staining is the most basic and widely used technique in histology, embryology, and pathology teaching and research.

[0048] The term "immunohistochemistry" refers to the application of the basic principle of immunology—the antigen-antibody reaction, that is, the principle of the specific binding of antigens and antibodies—to identify antigens (peptides and proteins) in tissue cells by making the chromogenic agent (fluorescein, enzyme, metal ion, isotope) labeled with antibodies show color through a chemical reaction. It is used to study the localization, qualitative and relative quantification of antigens. This technique is called immunohistochemistry or immunocytochemistry.

[0049] The invention generally described herein will be more readily understood by referring to the following examples, which are provided by way of illustration and are not intended to limit the invention. Furthermore, unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the raw materials, reagents, and other materials used in the following examples are commercially available products.

[0050] We constructed hPSC-induced whole-brain organoids (denoted as WT-organoids) using the Lancaster laboratory method. Furthermore, to improve the sensitivity of detecting abnormal cell proliferation, we constructed TP53. - / - / PTEN - / - Brain tumor organoids (GBM-organoids) derived from hPSCs (TP53 and PTEN double knockout) were used. The stimulation of the tumor microenvironment within these organoids promotes cell proliferation, aiming to improve the sensitivity of the safety testing platform in detecting abnormal cells. Following standard animal tumorigenesis experimental procedures, stem cells, induced-differentiation midbrain dopamine cells (mDA cells), early dopamine progenitor cells (dopamine cells differentiated to day 15), and dopamine cells from a mixture of stem cells were injected into the brain organoids and mouse striatum, respectively. Figure 1 As shown.

[0051] In this invention, the stem cells used are hPSCs. Those skilled in the art will expect that other stem cells that can differentiate into dopaminergic cells can also be used in this invention.

[0052] 1. Dopaminergic cell differentiation induction

[0053] Dopaminergic cell differentiation was induced according to the Kwang-Soon Kim laboratory protocol (Song et al. J Clin Invest. 2020). Early differentiation stage: differentiation was carried out in 10-cm culture dishes coated with Matrigel (Corning); on day 15 of differentiation, cells were digested with Accutase (Sigma) and passaged into Poly-L-ornithine / Fibronectin / Laminin (Sigma) coated culture dishes; Late differentiation stage: the basal medium was DMEM / F12 (Thermofisher), and the small molecule compounds were BDNF (Peprotech), GF-β3 (Peprotech), dbcAMP (Sigma), ascorbic acid (Sigma), TGF-β3 (Peprotech), and DAPT (Targetmol).

[0054] 2. Brain organoid differentiation induction

[0055] Brain organoid differentiation was induced according to the Lancaster laboratory protocol using commercially available whole-brain organoid culture medium (STEMCELL Technologies). 9,000 stem cells were added to each well of an ultra-low adsorption 96-well plate; on day 5, embryoid bodies (EBs) from the 96-well plates were transferred to ultra-low adsorption 24-well plates for further differentiation induction; on day 7, each EB was coated with Matrigel; starting on day 10 of differentiation, the culture medium was changed to maturation medium, and subsequent culture was performed using a shaker. The hPSCs for whole-brain organoids were unmutated, and the hPSCs for brain tumor organoids were TP53 cells constructed using CRISPR / Cas9 technology. - / - / PTEN - / - hPSCs.

[0056] 3. Fluorescent protein labeling of test cells

[0057] Lentiviral cells (self-constructed) carrying RFP or Luciferase fluorescent protein were added to stem cells to be tested, midbrain dopamine cells (mDA cells) induced from stem cells, early dopaminergic precursor cells induced from stem cells (dopaminergic cells on day 15 of differentiation), and dopaminergic cells of mixed stem cells. After 24 hours, the infection efficiency was observed by fluorescence microscopy.

[0058] Fluorescent proteins can be selected from one or more of the following, depending on the actual requirements: RFP, OFP, YFP, GFP, CFP, BFP, Luciferase, and tdTomato signal.

[0059] 4. Brain organoid cell injection

[0060] The fluorescently labeled cells were routinely digested and collected in centrifuge tubes. 6 μL of cell suspension was aspirated using a Hamilton microinjection needle, which was then fixed to an automated injection pump. The brain organoids were gently aspirated from the dish using a cut pipette tip and placed in a culture dish, with culture medium added to the surface. The injection pump automatically injected the cells at a rate of 1 μL / min. After injection, the brain organoids were returned to the culture dish using a cut pipette tip, and fresh culture medium was added. The dish was then returned to a CO2 incubator.

[0061] 5. Stereotactic transplantation of mouse brain

[0062] Dopaminergic cells were injected into the left striatum of mice using stereotactic surgery at coordinates: AP +0.5mm, ML -1.8mm, DV -3.2mm. Anesthesia was administered with isoflurane. The cell density was 50,000 / μL, with 2μL injected at a rate of 0.4μL / min. After injection, the cells were left in the mouse brain for 5 minutes, followed by slow withdrawal over another 5 minutes. Post-operatively, the mice were given 0.9% saline solution.

[0063] The mice used were immunodeficient (NOD SCID) mice; all mice involved in this invention were NOD SCID mice.

[0064] 6. Brain organoid sections, mouse brain sections, H&E staining, immunohistochemistry, and immunofluorescence.

[0065] After a certain period of time following the injection of the target cells, routine H&E staining, immunohistochemistry, and immunofluorescence were performed on brain organoids and mouse brains.

[0066] The specific implementation method is as follows:

[0067] Example 1. Dopaminergic cells can maintain normal growth and differentiation in brain organoids.

[0068] After injection of directed-induced differentiated dopaminergic cells into brain organoids and NOD SCID mice: (1) Morphology: Dopaminergic cells were scattered in brain organoids, and some neuronal axons extended extensively into the surrounding area of ​​the brain organoids after 3-4 weeks. (2) Efficacy: 4 weeks after injection, 80% of the injected dopaminergic cells expressed the dopaminergic precursor marker FOXA2 in brain organoids, and 30% of the cells expressed the mature dopaminergic neuron marker TH. (3) Proliferative capacity: The injected dopaminergic cells maintained a low level of proliferation similar to that in whole-brain organoids and mouse brains, even in brain tumor organoids. The above suggests that dopaminergic cells can maintain normal growth and differentiation in brain organoids.

[0069] like Figure 2 As shown, dopaminergic cells injected into brain organoids were labeled as RFP (A); bright-field and in vivo imaging of dopaminergic cells in whole-brain organoids (B) and brain tumor organoids (C) at 2 and 4 weeks post-injection; scale bar: 1000 μm for low-power scale and 200 μm for high-power scale; D. Immunofluorescence of FOXA2, TH, and Ki67 after injection into brain organoids; scale bar: 1000 μm for high-power scale and 80 μm for low-power scale; E. Immunofluorescence of hNuMA, FOXA2, TH, Ki67, and SOX1 after transplantation of dopaminergic cells into NOD SCID mice; scale bar: 80 μm; F. Statistical graph of immunofluorescence markers among whole-brain organoids, brain tumor organoids, and mice.

[0070] Example 2: Stem cells can maintain high proliferative capacity and stemness in brain organoids.

[0071] Stem cell transplantation is often used as a positive control in traditional animal experiments. In brain organoids: (1) Morphology: After injection into brain organoids, stem cells are mainly distributed at the injection site, exhibiting a clonal aggregation growth pattern. Over time, stem cells continue to grow and begin to infiltrate adjacent areas of the brain organoid. (2) Proliferative capacity: Two and four weeks after injection, stem cells in brain organoids expressed approximately 90% and 40% of the proliferation marker Ki67, respectively, showing proliferative capacity similar to that of the mouse brain. (3) Stemness: Compared to the mouse brain, the expression of the stem cell marker OCT4 was significantly upregulated two and four weeks after stem cell injection into brain organoids. (4) Histology: Densely aggregated, deeply stained cells were clearly visible in the brain organoids. Immunohistochemistry showed that the injected stem cells had the ability to invade adjacent brain organoid regions, indicating their high proliferative capacity. In summary, stem cells can maintain high proliferative capacity and stemness in brain organoids.

[0072] like Figure 3 As shown, Figure 3 The present invention describes the ability of stem cells to maintain high proliferative capacity and stemness in brain organoids; RFP-labeled stem cells to be injected (A); bright-field and in vivo imaging images of stem cells injected into whole-brain organoids (B) and brain tumor organoids (C) 2 and 4 weeks later; low-magnification scale bar: 1000 μm, high-magnification scale bar: 200 μm; D. Immunofluorescence images of OCT4, Nestin, and Ki67 after stem cells are injected into brain organoids; high-magnification scale bar: 1000 μm, low-magnification scale bar: 80 μm; E. Immunofluorescence images of hNuMA, OCT4, Nestin, and Ki67 after stem cells are transplanted into mice; scale bar: 80 μm; F. Quantitative statistical graph of immunofluorescence markers among whole-brain organoids, brain tumor organoids, and mice.

[0073] like Figure 4As shown, A. H&E staining images after stem cells are injected into brain organoids and mice; high magnification scale bar: 1000 μm, low magnification scale bar: 100 μm; B. Immunohistochemical image after stem cells are injected into brain organoids (showing RFP); scale bar: 100 μm.

[0074] Example 3: Residual stem cells can be sensitively detected in brain organoids.

[0075] To test the sensitivity of brain organoids as a safety assessment tool, we further conducted a dilution experiment (spiking experiment). We labeled stem cells with Luciferase and dopaminergic cells with RFP, and injected dopaminergic cells mixed with 10% or 1% (cell count) stem cells into brain organoids and NOD SCID mice. (1) Morphology: Dopaminergic cells mixed with 1% and 10% stem cells could survive in all brain organoids, and the Luciferase signal intensity gradually increased. Among them, brain tumor organoids had a stronger ability to detect residual stem cells; (2) Proliferative capacity: Brain tumor organoids could make the stem cells mixed with dopaminergic cells exhibit higher proliferative capacity; (3) Stem cell nature: Brain tumor organoids could make the stem cells mixed with dopaminergic cells exhibit higher pluripotency; (4) Histology: Deeply stained cells were visible in brain organoids. Immunohistochemistry also showed that residual stem cells could spread and invade surrounding brain organoids. The above results indicate that even when dopaminergic cells contain a low proportion of stem cells, brain organoids can detect and maintain stem cell growth, suggesting that brain organoids have high sensitivity in detecting residual stem cells. Among them, brain tumor organoids have an even stronger ability to detect residual stem cells.

[0076] like Figure 5 As shown, dopaminergic cells were labeled as RFP, while stem cells were labeled as Luciferase; a dopaminergic cell suspension containing 10% or 1% stem cells was prepared and injected into brain organoids (A); Luciferase signal was dynamically observed at 2, 4, 8 and 12 weeks after injection (B); C. Quantitative statistical graph of relative Luciferase expression among different groups.

[0077] like Figure 6 As shown, A. Immunofluorescence images of OCT4, FOXA2, and Ki67 after dopaminergic cells mixed with residual stem cells were injected into brain organoids; scale bar: 1000 μm for high magnification and 80 μm for low magnification; B. Immunofluorescence images of hNuMA, FOXA2, TH, Ki67, and SOX1 after dopaminergic cells mixed with residual stem cells were transplanted into mice; scale bar: 80 μm; C. Quantitative statistical graph of immunofluorescence markers among whole-brain organoids, brain tumor organoids, and mice.

[0078] like Figure 7As shown, A. H&E staining images of dopaminergic cells mixed with residual stem cells after injection into brain organoids and NOD SCID mice; high magnification scale bar: 1000 μm, low magnification scale scale bar: 100 μm; B. Immunohistochemical staining images of dopaminergic cells mixed with residual stem cells after injection into brain organoids; scale bar: 100 μm.

[0079] Example 4: High proliferation and growth of early dopaminergic precursor cells in brain tumor organoids

[0080] Immature early neural progenitor cells in cell products can increase the risk of tumor formation. For example, approximately 40% of rat brains receiving early dopaminergic progenitor cells at day 15 of differentiation exhibit rosette-like structures. To verify the fate changes of early dopaminergic progenitor cells in brain organoids, we used day 15 of differentiation dopaminergic cells as early dopaminergic progenitor cells and injected them into brain organoids and NOD SCID mice. The results showed that: (1) Efficacy: Approximately 75% of the cells expressed the dopaminergic progenitor marker FOXA2 2 weeks after injection. (2) Proliferative capacity: Compared with whole brain organoids and rat brains, the proliferative capacity of early dopaminergic progenitor cells in brain tumor organoids was significantly enhanced. This suggests that early dopaminergic progenitor cells exhibit high proliferative growth in brain tumor organoids.

[0081] like Figure 8 Figure 1 shows bright-field and in vivo imaging images at 2 and 4 weeks after dopaminergic cells were injected into whole-brain organoids (A) and brain tumor organoids (B) on day 15 of differentiation; scale bar: 1000 μm for low-power scale and 200 μm for high-power scale. Figure 2 shows immunofluorescence images of FOXA2, TH, and Ki67 after early dopaminergic precursor cells were injected into brain organoids; scale bar: 1000 μm for high-power scale and 80 μm for low-power scale. Figure 3 shows immunofluorescence imaging images of hNuMA, FOXA2, TH, Ki67, and SOX1 after early dopaminergic precursor cells were transplanted into NOD SCID mice; scale bar: 80 μm. Figure 4 shows a quantitative statistical graph of immunofluorescence markers among whole-brain organoids, brain tumor organoids, and mice.

[0082] Although exemplary embodiments of the invention have been shown in the foregoing disclosure, it should be noted that various changes and modifications can be made without departing from the scope defined by the claims. Furthermore, while elements of the invention may be described or claimed individually, it is also contemplated that multiple elements may be included, unless explicitly limited to a single element.

[0083] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for evaluating the safety of stem cell therapy based on brain organoids, characterized in that, Includes the following steps: S1. Constructing brain organoids; The brain organoids are one or both of whole-brain organoids and brain tumor organoids; S2. Prepare test cells and label them with fluorescent proteins; the test cells include one or more of the following: dopaminergic cells, stem cells, dopaminergic cells mixed with 1%-10% stem cells, and early dopaminergic precursor cells; S3. Inject the test cells labeled with fluorescent protein into brain organoids; S4. Analyze the test cells injected into brain organoids.

2. The method for evaluating the safety of brain organoid-based stem cell therapy according to claim 1, characterized in that, The brain tumor organoids were derived from TP53. - / - / PTEN - / - hPSCs.

3. The method for evaluating the safety of brain organoid-based stem cell therapy according to claim 2, characterized in that, The TP53 - / - / PTEN - / - hPSCs are obtained by knocking out the TP53 and PTEN genes in hPSCs using CRISPR / Cas9 technology.

4. The method for evaluating the safety of brain organoid-based stem cell therapy according to claim 1, characterized in that, The fluorescent protein in step S2 includes one or more of RFP, OFP, YFP, GFP, CFP, BFP, Luciferase, and tdTomato signal.

5. The method for evaluating the safety of brain organoid-based stem cell therapy according to claim 4, characterized in that, In step S2, the fluorescent protein is labeled by adding lentiviruses carrying RFP, OFP, YFP, GFP, CFP, BFP, Luciferase, and tdTomato signals to the cells to be tested and then culturing them.

6. The method for evaluating the safety of brain organoid-based stem cell therapy according to claim 1, characterized in that, Step S3 includes: routinely digesting and collecting the test cells labeled with fluorescent protein; drawing 3-10 μL of cell suspension using a microinjection needle; aspirating the brain organoid from the dish and placing it in a culture dish, adding culture medium; injecting the test cells at a rate of 0.5-2 μL / min; after injection, returning the brain organoid to the culture dish, replacing the culture medium, and returning it to the carbon dioxide incubator.

7. The method for evaluating the safety of brain organoid-based stem cell therapy according to claim 1, characterized in that, In step S4, the analytical methods for the test cells injected into the brain organoid include immunofluorescence analysis and histological analysis.

8. The method for evaluating the safety of stem cell therapy based on brain organoids according to claim 7, characterized in that, In the immunofluorescence analysis, the biomarker is preferably one or more of the following: stem cell biomarker OCT4, dopaminergic cell biomarker FOXA2, TH, and proliferation biomarker Ki67.

9. The method for evaluating the safety of brain organoid-based stem cell therapy according to claim 7, characterized in that, The preferred histological analysis is H&E staining and / or immunohistochemistry.

10. The method for evaluating the safety of brain organoid-based stem cell therapy according to claim 1, characterized in that, The stem cells mentioned are hPSCs.

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