Construction method of human microglial cell model carrying ENTPD1 gene mutation

Abstract

The invention discloses a construction method of a human microglial cell model carrying ENTPD1 gene mutation, and the human microglial cell model is prepared by the following steps: extracting PBMC from venous blood of a hereditary spasmodic paraplegia patient, then electrically transforming four factors of Oct4, Sox2, Klf4 and cMyc, and culturing in a Matrigel-coated culture dish for 15-20 days to obtain induced pluripotent stem cells; the induced pluripotent stem cells can form a unique human microglial cell model, and the human microglial cell model and the induced pluripotent stem cells are co-cultured to obtain an organoid, so that the analysis of a pathogenic molecular mechanism behind a disease risk gene is greatly facilitated, the defects of a rodent-derived microglial cell model are complemented, and the development of the rodent-derived microglial cell model is promoted. And the influence of the ENTPD1 on the related functions of the microglial cells is further discussed.

Description

Technical Field

[0001] The present invention relates to the field of cell culture, and in particular to a method for preparing induced pluripotent stem cells, and also to applications of induced pluripotent stem cells. Background Art

[0002] Hereditary spastic paraplegia (HSP) includes a group of clinically and genetically diverse neuromuscular degenerative diseases, characterized by progressive lower limb spasticity and weakness. Spastic paraplegia type 64 (HSP SPG64) is a complex hereditary spastic paraplegia (HSP) found in individuals diagnosed with a neurodegenerative disorder in recent years. Affected patients carry a homozygous mutation in the extracellular nucleoside triphosphate diphosphohydrolase 1 gene (ENTPD1).

[0003] CD39 is encoded by the ENTPD1 gene and is an extracellular nucleotidase that can bind to extracellular adenosine triphosphate (eATP) and convert it into extracellular adenosine (ADO) with a broad immunosuppressive effect. CD39 is a cell membrane protein and an extracellular nucleotide hydrolase encoded by the extracellular nucleotide triphosphate diphosphohydrolase (ENTPD) gene. It belongs to the ENTPD1 family and has ATPase and adenosine diphosphate (ADP) enzyme activities. It can hydrolyze extracellular ATP and ADP into adenosine monophosphate (AMP) and is the rate-limiting extracellular enzyme in the hydrolysis of extracellular ATP. CD39 is expressed in a variety of immune cells and non-immune cells (such as endothelial cells and fibroblasts), and CD39 and its downstream ADO receptors will also increase their expression levels under various stimuli including tissue damage, hypoxia and tissue remodeling. In the nervous system, CD39 is mainly highly expressed in microglia.

[0004] At present, the relevant research on ENTPD1 is mainly concentrated in tumors, blood, endocrine diseases, etc. The current research mostly uses the model of model organisms (such as mice) for related experiments. The modeling time is relatively long, the steps are relatively complex, and there are many uncertain factors, which to some extent affects the research progress and expected results.

[0005] Therefore, a humanized stem cell differentiation modeling method is urgently needed to study this disease. Summary of the invention

[0006] In view of this, one of the objects of the present invention is to provide a method for preparing induced pluripotent stem cells carrying ENTPD1 gene mutations; a second object of the present invention is to provide the use of the obtained induced pluripotent stem cells in the preparation of brain organoids or human microglia; a third object of the present invention is to provide a method for preparing brain organoids containing microglia.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] 1. A method for preparing induced pluripotent stem cells carrying ENTPD1 gene mutations, wherein the human microglial cell model is prepared by extracting PBMC from the venous blood of patients with hereditary spastic paraplegia, and then electroporating four factors, Oct4, Sox2, Klf4 and cMyc, and culturing them in a Matrigel-coated culture dish for 15 to 20 days to obtain induced pluripotent stem cells; the PBMC cells are preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: C2024402 and a preservation date of 2024.12.4, and are classified and named as human blood-derived induced pluripotent stem cells iPS-ENTPD1 Homo sapiens.

[0009] 2. Application of the obtained induced pluripotent stem cells in the preparation of brain organoids or human microglia.

[0010] Preferably, the method for preparing human microglia of the present invention is to induce microglia differentiation by using the induced pluripotent stem cell mTeSR1 / Matrigel culture system, and finally produce a mature human microglia model carrying the ENTPD1 gene mutation.

[0011] Preferably, the method for preparing the brain organoids is to separate induced pluripotent stem cells into single cells, inoculate them and supplement them with 50 μM rock inhibitor and 4 ng·ml -1 The cells were cultured in hESC medium containing FGF2 to promote embryoid body formation. On the 6th day, the embryoid bodies were transferred to a well plate containing neural induction medium to promote neural ectoderm formation. On the 11th day, the embryoid bodies were embedded in Matrigel and transferred to a culture dish containing brain organoid medium to promote neuroepithelium formation. On the 15th day, the culture medium was replaced with brain organoid medium, and the organoids were placed on an oscillator at 85 rpm and cultured until the 26th day to obtain brain organoids.

[0012] 3. The preparation method of brain organoids containing microglia is to mix NPC and PMP in a cell ratio of 7:3, aggregate them into precursor spheres with AggreWell, mix neuron culture medium and microglia culture medium in a ratio of 1:1 to obtain a mixed culture medium, add it to the precursor spheres, change three-quarters of the medium every 3 to 4 days, and culture for at least two months, and the brain organoids are basically formed.

[0013] The beneficial effects of the present invention are as follows: the present invention discloses a method for constructing a human microglial cell model carrying an ENTPD1 gene mutation, wherein PBMCs are extracted from the venous blood of a patient with hereditary spastic paraplegia using the human microglial cell model, and then four factors, Oct4, Sox2, Klf4 and cMyc, are electroporated and cultured in a Matrigel-coated culture dish for 15 to 20 days to obtain induced pluripotent stem cells; the obtained induced pluripotent stem cells can be induced to differentiate into brain organoids or human microglial cells, and the brain organoids and human microglial cells are co-cultured to obtain brain organoids carrying the ENTPD1 gene, which can be used as a hereditary spastic paraplegia disease model, and is of great significance for the study of the pathogenic mechanism and treatment method of hereditary spastic paraplegia diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0015] Figure 1 The cell morphology during the culture process;

[0016] Figure 2 Flow chart for microglial differentiation;

[0017] Figure 3 Flowchart for co-culturing microglia and forebrain organoids;

[0018] Figure 4 Flow chart for the differentiation and culture of forebrain organoids;

[0019] Figure 5 Mature NPCs and PMPs were co-cultured to generate brain organoids containing microglia.

[0020] Cell preservation

[0021] The human blood stem cell iPS-ENTPD1 in the present invention is deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: C2024402 and a deposit date of 2024.12.4, and is classified and named as human blood-derived induced pluripotent stem cell iPS-ENTPD1 Homo sapiens. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0023] Example 1: Extraction of PBMC from human blood and reprogramming to prepare iPSCs

[0024] 4-5 mL of fresh venous blood (in an anticoagulant tube) was extracted from the patient, and PBMC cells were separated from the blood by Lymphoprep gradient centrifugation. The cells were identified as PBMCs carrying the ENTPD1 gene mutation (c.1174delC, exon8, NM 001776, resulting in an amino acid change of p.Q392Sfs*6), named iPS-ENTPD1, ​​and the cells were preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: C2024402 and a preservation date of 2024.12.4. The cells were classified and named human blood-derived induced pluripotent stem cells iPS-ENTPD1 Homo sapiens. After 5-7 days of culture, four Yamanaka factors, Oct4, Sox2, Klf4 and cMyc, were electroporated, and the cells were seeded in a culture dish coated with Matrigel (Corning 354230). After 15-20 days of electroporation, iPSC (induced pluripotent stem cell) clones appeared. The cell morphology on the 8th, 15th and 20th days of culture was as follows Figure 1 As shown, the clones are dense and undifferentiated.

[0025] Multiple iPSC clones were selected and analyzed for karyotype, stem cell stemness, etc.

[0026] Example 2: Stem cell culture and induction of microglial differentiation

[0027] The high-quality clones of the embodiment were selected and cultured normally (37°C cell culture incubator) with mTeSR1 / Matrigel culture system containing BMP4, Activin-A, VEGF, TPO, SCF, IL-3 and IL-6 to start microglial differentiation, and then cultured in the presence of M-CSF, IL-34 and TGFβ1, and then cultured under conditions containing CD200 and CX3CL1, and finally mature human iPSC-derived microglia-like cells (hMGLs) were generated. During the culture process, flow cytometry was used to screen CD43 + Positive HPCs have cell morphology during culture. Figure 2 As shown in the flow chart, Figure 3 As shown, the results showed that the clones were dense and undifferentiated, and mature microglia had pseudopodia.

[0028] Example 3: Stem cell differentiation brain organoids protocol

[0029] Human iPSCs were dissociated into single cells using Stempro accutase, and 9,000 iPS cells from each organoid were seeded into each well of an ultra-low attachment 96-well plate in a 96-well plate supplemented with rock inhibitor (50 μM) and FGF2 (4 ng ml -1) in hESC medium (Catalog #85850) to promote embryoid body formation. On day 6, the embryoid bodies were transferred to an ultra-low attachment 24-well plate containing neural induction medium to promote neural ectoderm formation. On day 11, the embryoid bodies were embedded in Matrigel and transferred to a 10 cm culture dish containing brain organoid medium (B-27 supplemented medium without vitamin A, product number 12587010) to promote neuroepithelium formation. On day 15, the culture medium was replaced with brain organoid medium (B-27 supplemented medium with vitamin A), and the organoids were placed on an oscillator at 85 rpm and cultured until day 26 to obtain brain organoids. During the culture process, in order to maximize nutrient absorption and further mature each organoid. The culture medium is replaced every three days to ensure optimal utilization of nutrients and maturation of organoids to ensure their long-term survival. The process is shown in the figure Figure 3 shown.

[0030] Example 4: Co-culturing forebrain organoids and microglia

[0031] The 150,000 hMGLs cultured in Example 2 (day 26) and the brain organoids obtained in Example 2 (day 26) were incubated in 1 ml of brain organoid culture medium (containing B-27 supplemented culture medium, vitamin A and 100 ng ml -1 CSF-1) was co-cultured to reduce the adhesion of hMGL. This time point was called day 0. On day 2, another 1 ml of brain organoid culture medium (containing B-27 supplemented medium, vitamin A, and 100 ng ml -1 CSF -1 ). Starting from the 5th day, half of the culture medium was replaced every three days for 18 days. The culture process was as follows Figure 4 shown.

[0032] Example 5: Direct differentiation of organoids containing microglia

[0033] iPSCs differentiate into NPCs (neural progenitor cells) and PMPs (macrophage progenitor cells), which are the precursors of neurons, astrocytes, and microglia, respectively. The two are mixed at a cell ratio of 7:3 and aggregated into precursor spheres using AggreWell. Mix neuronal culture medium and microglial culture medium in a 1:1 ratio to obtain a mixed culture medium, add it to the precursor spheres, change the medium by three quarters every 3 to 4 days, and culture for at least two months. Brain organoids are basically formed ( Figure 5 ).

[0034] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A method for preparing induced pluripotent stem cells carrying ENTPD1 gene mutation, characterized in that: The human microglial cell model is prepared by extracting PBMC from the venous blood of patients with hereditary spastic paraplegia, and then electroporating four factors, Oct4, Sox2, Klf4 and cMyc, and culturing them in a Matrigel-coated culture dish for 15 to 20 days to obtain induced pluripotent stem cells; the PBMC cells are preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: C2024402, a preservation date of 2024.12.4, and are classified and named as human blood-derived induced pluripotent stem cells iPS-ENTPD1 Homo sapiens.

2. Use of the induced pluripotent stem cells obtained according to claim 1 in the preparation of brain organoids or human microglia.

3. The use according to claim 2, characterized in that: The method for preparing human microglia is to induce microglia differentiation by using an induced pluripotent stem cell mTeSR1 / Matrigel culture system, and finally generate a mature human microglia model carrying the I344E-SPAST gene mutation.

4. The use according to claim 2, characterized in that: The method for preparing the brain organoids is to separate induced pluripotent stem cells into single cells, inoculate them in a solution supplemented with 50 μM rock inhibitor and 4 ng ml -1 The cells were cultured in hESC medium containing FGF2 to promote embryoid body formation. On the 6th day, the embryoid bodies were transferred to a well plate containing neural induction medium to promote neural ectoderm formation. On the 11th day, the embryoid bodies were embedded in Matrigel and transferred to a culture dish containing brain organoid medium to promote neuroepithelium formation. On the 15th day, the culture medium was replaced with brain organoid medium, and the organoids were placed on an oscillator at 85 rpm and cultured until the 26th day to obtain brain organoids.

5. A method for preparing a brain organoid containing microglia, characterized in that: NPCs and PMPs were mixed at a cell ratio of 7:3, aggregated into precursor spheres using AggreWell, and mixed with neuronal culture medium and microglial culture medium at a ratio of 1:1 to obtain a mixed culture medium, which was added to the precursor spheres. The medium was replaced by three-quarters every 3 to 4 days and cultured for at least two months until brain organoids were basically formed.

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