CN111334477A

CN111334477A - Cerebral cortex organoid of alzheimer's disease and obtaining method and application thereof

Info

Publication number: CN111334477A
Application number: CN202010225151.2A
Authority: CN
Inventors: 范文娟; 邓锦波; 陈旭东
Original assignee: Luohe Medical College
Current assignee: Luohe Medical College
Priority date: 2020-03-26
Filing date: 2020-03-26
Publication date: 2020-06-26

Abstract

The invention relates to the technical field of biomedicine, in particular to an Alzheimer disease cerebral cortex organoid and an obtaining method and application thereof, which utilizes lentivirus infection to transfer APP and PS1 mutant genes into mouse induced pluripotent stem cells and establish an iPSCs line capable of stably transferring the APP and PS1 mutant genes. And then establishing a mature and stable cerebral cortex organoid culture system of the mouse Alzheimer disease through 3D culture, which not only can help researchers to further understand the mechanism of human development, but also can be used as a disease model or a drug screening platform.

Description

Cerebral cortex organoid of alzheimer's disease and obtaining method and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to an Alzheimer disease cerebral cortex organoid and an obtaining method and application thereof.

Background

Organoid culture (organoid culture) is an artificial tissue culture technique that has recently emerged in recent years with rapid development in the stem cell field. It is a complex, living tissue-like structure that is generated from stem cells from single cells, mainly by means of 3D culture. In the application aspect, the method relates to the field of artificial organs, and researches on living tissue occurrence, mechanism of disease formation, drug experiments, tissue replacement therapy and the like.

Induced pluripotent stem cells (ipscs) are a type of pluripotent stem cells obtained by reprogramming differentiated mature somatic cells by transferring some stem cell transcription factors. It is similar to embryonic stem cells in biological properties and can differentiate into somatic cells in multiple directions. Compared with embryonic stem cells, iPS cells are convenient to obtain and wide in source, do not have the problems of immunological rejection, ethical morality and the like, and are the most ideal seed cells for organoid culture.

Alzheimer's Disease (AD) is one of the most common neurodegenerative diseases with progressive progression, and mutations in Amyloid Precursor Protein (APP) and Presenilin (PS) genes are major factors that contribute to the development of familial AD.

For a long time, the AD transgenic animal model is a main research tool for exploring the pathophysiological mechanism of AD, but the AD transgenic animal model has various problems of high modeling difficulty, long feeding period, mutual interference among multiple genes in vivo and the like, so that the research on the occurrence of a certain key pathological event is difficult. At present, in domestic research, no research report related to the culture of cerebral cortex organoids, particularly the culture of cerebral organoids of Alzheimer disease is seen.

Disclosure of Invention

In view of the above, the invention obtains the iPSCs line stably transferring APP and PS1 mutant genes by overexpressing APPSwe/Ind (APP) and PSEN1(PS1) mutant genes in iPSCs, and establishes an experimental model capable of rapidly generating AD pathological phenotype by culturing AD cerebral cortex organs with the iPSCs line.

The invention provides a method for obtaining cerebral cortex organoids of Alzheimer's disease, which comprises the following steps:

s1, transferring the APPSwe/Ind-PSEN1 double mutant genes into mouse iPSCs by a lentivirus transfection mode to obtain the iPSCs containing the double mutant genes;

s2, carrying out subculture on the iPSCs containing the double mutant genes, and removing feeder layer cells by using a differential adherence method to obtain purified iPSCs;

s3, inoculating the purified iPSCs on a cell culture plate a, culturing in a first culture medium, changing liquid for half a day every 2 days, culturing for 4-6 days, transferring an embryoid body formed by the iPSCs to a cell culture plate b, culturing in a second culture medium, changing liquid once every two days, coating a semitransparent neuroepithelial bud-shaped protrusion extending out of the periphery of the embryoid body when culturing for 6-8 days, changing a third culture medium for low-speed shaking culture, changing liquid once every two days, continuously culturing for 60-65 days, collecting a specimen, and obtaining the cerebral cortex organs of the Alzheimer disease;

the first culture medium is: LIF-free iPSCs serum-free medium;

the composition comprises Knockout DMEM (Gibco, 10829018), KSR (Gibco, 10828028) with the mass fraction of 20%, 100X × L-GlutaMax (Gibco, 3505006), 100 × NEAA (Gibco, 11140-.

The second culture medium is: a neuroepithelial differentiation medium;

the components are DMEM-F12(Gibco, 11330032), 100 × N2 supplement (Gibco, 17502-;

the third culture medium is: cerebral cortex differentiation medium;

the ingredients are DMEM/F12(Gibco, 11330032), Neurobasal medium (Gibco, 21103-049), 50 × B27(Gibco, 17504-044), 100 × N2 supplement (Gibco, 17502-048), 100 × L-GlutaMax (Gibco, 3505006), 100 × NEAA (Gibco, 11140-050), 100 × P/S (Gibco, 15140-122), 2 mu g/LMouse IGF-I (R & D, 791-MG).

Further, in S3, the concentration of purified iPSCs was 1 × 10⁴One by 100. mu.l.

Further, in S3, the cell culture plate a was a low-adhesion U-bottom 96-well plate, and the cell culture plate b was a low-adhesion 6-well plate.

Further, in S3, Rock inhibitor Y27362 was added to the first medium.

Further, the final concentration of the inhibitor Y27362 was 50. mu.M.

Further, in S3, the translucent neuroepithelial bud-like protrusion extending from the periphery of the embryoid body is coated with Matrigel.

Further, in S3, the culture conditions were all 37 ℃ and 5% by volume of CO₂And balancing humidity.

The invention also provides the cerebral cortex organoid of the Alzheimer disease obtained by the method.

Furthermore, the application of the cerebral cortex organoid of the Alzheimer disease as an Alzheimer disease model.

Furthermore, the application of the cerebral cortex organoid of the Alzheimer disease as a screening platform of the Alzheimer disease treatment drug.

Compared with the prior art, the invention has the following beneficial effects:

the invention utilizes an in vitro 3D culture technology to transfer two AD mutant genes of APP and PS1 into iPS cells, and forms AD brain organoids through suspension culture, thereby providing important reference value for researching regeneration repair and function reconstruction of central nervous system injury. In addition, since organoid culture can simulate a part of the structure and function of a real organ, it not only helps researchers understand the mechanism of human development, but also can be used as a disease model or a drug screening platform, and is expected to become a source of organ transplantation. Although the current organoid culture technology is not mature, the organs and tissues from which ES cells or iPS cells are obtained in a three-dimensional culture environment are expected to be more and more similar to the organs from which in vivo development is derived, and the organoid culture technology is likely to be widely applied to more fields as a good carrier.

Drawings

FIG. 1 is a schematic diagram of construction of an APP and PS1 mutant gene lentiviral vector, wherein A: APP695 refers to a gene sequence carrying an APPSwe/Ind mutant; b is_：PSEN1 refers to the gene sequence carrying the PSEN1 Δ E9 mutant; c: and constructing a finished plasmid map.

FIG. 2 results of flow cytometric fluorescence sorting of transfected cells.

FIG. 3 shows the Western Blot detection results of the mutant genes and their cleavage products.

FIG. 4 shows the level of A β 1-40 and A β 1-42 secreted extracellularly by iPSCs transformed with AD mutants.

FIG. 5 shows embryoid bodies formed by spontaneous differentiation of APP PS1 mutant iPSCs.

FIG. 6 shows the development process of brain cortex organoids formed by AD mutant iPSCs and normal iPSCs.

FIG. 7 shows the extracellular secretion levels of A β 1-40, A β 1-42 and Tau protein which is hyperphosphorylated.

FIG. 8 shows the expression levels of AD brain organoid mutant genes APP and PS1 and pathological marker proteins P-Tau and A β in DIV 63.

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.

Example 1

Method for obtaining cerebral cortex organoid of alzheimer disease

The method specifically comprises the following steps:

s1, constructing iPSCs containing double mutant genes

1. Construction of Lentiviral expression vectors

pCAX APPSwe/Ind: gifts were given by Dennis Selkoe and Tracy Young-Pearse (Addgene plasmid # 30145).

pCMV6 PSEN1 (pLenti-C-PSEN 1-mGFP, infra): from OriGene technologies (RC 216443; GeneBank accession No.: NM-000021).

Lentiviral packaging plasmids: pLVX-IRES-mCherry, pLVX-IRES-ZsGreen1, and their helper plasmids pSPAX2, pMD2.G were purchased from Hunan Youbao biotech.

First, plasmids pCAX APPSwe/Ind and pLenti containing two mutant genes were usedTransformation and amplification of-C-PSEN 1-mGFP, transformation of the correctly sequenced gene product, amplification, plasmid extraction, and separate application of EcoRI and XhoI to two T-vectors (for

T Easy vector, purchased kit, original name:

t Easy Vector Systems (Promega, A1360)), by double digestion.

Then enzyme digestion products are recovered and purified, and then are respectively constructed on pLVX-IRES-ZsGreen1 and pLVX-IRES-mCherry lentiviral vectors in a homologous recombination mode to obtain pLVX-APP-IRES-ZsGreen1, pLVX-APP-PS1-IRES-ZsGreen1 and pLVX-PS 1-IRES-mChery lentiviral expression plasmids (the constructed plasmid map is shown in figure 1). And (5) after the vector is successfully constructed, delivering the vector to a sample for sequencing. Primer synthesis and sequencing are completed by the department of Okinawa Okagaku Biotech, Hunan.

2. Viral packaging and infection of iPS cells

(1) The 293T cells were thawed to a 10mm culture dish and when they were grown to 70-80% abundance, Lipofectamine was used^TM3000 the following combinations of lentiviral expression plasmids containing mutated genes and their helper plasmids (pSPAX2/Pmd 2.G) were transfected separately.

Specific transfection procedures were according to Lipofectamine^TM3000 the specification.

(2) The next day, the medium was changed for each group of 293T cells after transfection, and the supernatant was retained.

(3) On day 3, 293T medium was collected, along with yesterday's collection, filtered through a 0.22 μm filter and polybrene was added to a final concentration of 8 μ g/ml, which was the viral supernatant. 293T cells were continuously replaced with fresh medium for secondary infection.

(4) MEFCs in mouse iPSCs are removed by a differential adherence method, and after centrifugation, the MEFCs are resuspended by a small amount of iPSCs culture medium and inoculated into a 6mm culture dish, wherein each well contains 0.5ml of resuspension. Adding 2ml of corresponding virus supernatant into each well of iPS-GA, iPS-RP, iPS-GAP and iPS-G, iPS-R according to different groups; 2ml of each of the A-G supernatant and the P-mCH was added to the iPS-AP group. The culture was carried out overnight. Additionally, MEFCs are revived for use.

(5) The next day, virus supernatants were removed, the iPSCs medium was replaced, and culture continued on feeder cells. After 5 days, passage is carried out, and virus secondary infection is carried out, and the method is the same as the above. Finally, the virus infection efficiency is analyzed through the expression of the fluorescent marker.

Identification of APP and PS1 mutant iPSCs

1) GFP or mCherry channels were used to detect expression of transfected genes in individual cells and to sort out the corresponding fluorescent cells by flow cytofluorescent Sorting (FACS) technique. The results are shown in FIG. 2:

in fig. 2, fig. 2A: FACS sorting the iPSCs carrying ZsGreen1 green fluorescent lentiviral vector markers;

FIG. 2B: FACS sorting carried mCherry red fluorescence, fig. 2C: the iPSCs carrying ZsGreen1 green fluorescence and carrying mCherry red fluorescence labeled AD mutant genes are subjected to amplification culture, and purified and enriched fluorescent cells can be observed after passage. (Scale 40 μm)

(2) Western Blot is used for detecting the expression of the mutant gene and the cleavage product thereof. The results are shown in FIG. 3:

in FIG. 3, the protein expression levels of full-length APP (FL-APP) and soluble fragments sAPP α and sAPP β in the control group (iPS-G) and the AD mutant iPSCs (iPS-GA, iPS-GAP) treated by Western blot, the protein expression levels of full-length APP (FL-APP) and soluble fragments sAPP α and sAPP β in the AD mutant iPSCs (iPS-GAP ) transferred into the control group (iPS-R) and the AD mutant iPSCs (iPS-RP, iPS 3578 (FL-APP) and the protein expression levels of PS 1N-terminal (PS1-NTF) and PS 1C-terminal (PS1-CTF) generated by endoproteinase digestion are detected in Western blot 3A, the statistical analysis is performed by using GraphPad Prism software, the gene fragments of the iPS-GAS group and the full-length APP α and the full-length fragments of the AD mutant iPS 2 (iPS-PAPs-GAP) are significantly increased by < 0.05.05 < PP < SP 23.01.01.01, and < SP-7.05.05.01 < SP-7.

(3) The ELISA kits of A β 40 and A β 42 are used for detecting the levels of A β 1-40 and A β 1-42 secreted outside cells of iPSCs transferred into the AD mutant, and the results are shown in FIG. 4:

FIGS. 4A-C in FIG. 4 show the extracellular secretion levels of A β 1-40 and A β 1-42 and the ratio of A β 1-42 to A β 1-40 of undifferentiated AD mutant iPSCs, respectively, as detected by ELISA kits A β 40 and A β 42, the data results are expressed as Mean. + -. standard error (Mean. + -. SEM), the statistical treatment is by one-way anova, the analysis of significant difference is by t-test, p is 0.05, p is 0.01, p is 0.001.

FIG. 5 shows embryoid bodies formed by spontaneous differentiation of APP PS1 mutant iPSCs. FIGS. 5A-E: except the iPS-GAP group, the iPSCs dissociated from other groups can be rapidly aggregated within 30 min; FIGS. 5F-J: morphology of each set of iPSCs aggregates was cultured in vitro the next day. (Scale: A-J ═ 40 μm)

The results show that the iPSCs transferred into the APPSwe-PSEN1 double mutant gene have more obvious pathological phenotypic characteristics and neural differentiation disorder. In the embodiment, iPSCs with APPSwe/Ind-PSEN1 double mutant genes are transferred to culture AD cerebral cortex organoids, and iPSCs without mutant gene vectors are transferred to culture control cerebral cortex organoids.

S2, removing feeder layer cells of the two groups of iPSCs after normal passage by using a differential adherence method to obtain purified iPSCs;

s3, purified iPSCs were dropped into a low-adhesion U-bottom 96-well plate at a concentration of 1 × cells/100. mu.l in a LIF-free iPSCs serum-free medium (composition: knockoutDMEM (Gibco, 10829018); 20% KSR (Gibco, 10828028); 100X × L-GlutaMax (Gibco, 3505006); 100 × NEAA (Gibco, 11140-Busy 050); 1000X × ME 2-ME (10mM) (2-Mercaptoethanol, Sigma, M3148)) and Rock inhibitor Y27362 (final concentration: 50. mu.M) were added to maintain the viability of the iPS, and then placed in a 37 ℃, 5% CO2, humidity-balanced CO2 incubator, half a day after 4 days of culture, the embryos formed by two sets of iPSCs were transferred to a low-adhesion U-bottom 96-well plate, and cultured in a neural cell culture medium (Gibco- ×, ×) in a neural differentiation medium (Gibco-100. mu.M) 7378-PL, and then transferred to a neutral medium (

Gibco

3, 6858, 3, and5006)；100×NEAA(Gibco，11140-050)；Mouse Noggin(R&d, 1967-NG-02)) was started and the culture was changed every two days under the conditions of 37 ℃ and 5% CO₂(ii) a The low adhesion culture plate can keep the suspension culture state all the time, which is very important for the culture of cerebral cortex organoids.

The semi-transparent neuroepithelial bud-like protrusion around the embryoid body can be observed under a light microscope after the neuroepithelial differentiation medium is cultured for about one week, at the moment, Matrigel is used for coating, and cerebral cortical differentiation medium (the components are DMEM/F12(Gibco, 11330032); Neurobalalmedium (Gibco, 21103) 049); 50 × B27(Gibco, 17504) 044); 100 × N2 deletion (Gibco, 17502) 048); 100 × L-Glutamax (Gibco, 3505006); 100 × NEAA (Gibco, 11140) 050); 100 × P/S (Gibco, 15140) 122); 2 ug/LMse IGF-I (R & D, 791-MG) is placed on a micro shaking plate bed, and cultured in a 5% CO2 and 5% humidity balanced culture box at 37 deg.C, the culture medium temperature is further, and the culture medium is subjected to low humidity culture, observed after the organ culture medium is shaken for one time, and the culture medium is subjected to low humidity culture, and the tissue culture medium is observed for further treated by shaking for one time, so as the Alzheimer' S culture medium, so as the tissue culture medium for observing.

Example 2

Pathological phenotype of cerebral cortex organoid and experiment of neurogenesis thereof

In FIG. 6, FIGS. 6A-H: in order to form cerebral cortex organoids by the AD mutant iPSCs and normal iPSCs under the light microscope, a plurality of neuroepithelial buds (G, ×) are easily generated in the AD cerebral cortex organoids. (Scale: A, B, E, F ═ 40 m; C, D, G, H ═ 20m)

Organoid culture supernatants were collected at 35 and 63 days of in vitro brain organoid culture (Day in vitro, DIV35, DIV63), and the levels of extracellular A β 1-40, A β 1-42, and hyperphosphorylated Tau protein (P-Tau) secretion were measured by ELISA, as shown in FIG. 7, that the APPSwe/Ind and PSEN1 double mutations contributed to the pathological overproduction of A β 40, A β 42, and P-Tau proteins.

In FIG. 7, FIGS. 7A-C show a significant increase in both A β 1-40 and A β 1-42, and a significant increase in the A β 42/A β 40 ratio in AD brain organoid culture medium compared to control, FIG. 7D shows extracellular P-Tau secretion measured by ELISA, no significant change in P-Tau of AD brain organoids compared to control in DIV35, and a significant increase in P-Tau in culture medium in DIV63, the data were obtained from 3 independent tests, and the results were expressed as Mean + -standard error (Mean + -SEM), and were statistically processed by one-way anova with t-test for significant difference analysis, P <0.05, P <0.01, P < 0.001.

In FIG. 8, the expression levels of AD brain organoid mutant genes APP and PS1 and pathological marker proteins P-Tau and A β were significantly increased in Western blot detection of DIV63 in FIG. 8A, the expression levels of AD brain organoid FL-APP and FL-PS1 were significantly increased in comparison with the control group (P <0.01 and P <0.001, respectively) in FIG. 8B, the expression levels of P-Tau and A β proteins were significantly increased in comparison with the control group (P <0.001 and P <0.05, respectively) in AD brain organoid P-Tau and A β, the expression levels of P-Tau and A β proteins were significantly decreased after DAPT treatment (P <0.05), the data were obtained from 3 independent tests, the results were expressed as Mean. + -. standard error (Mean. + -. SEM), statistical treatment was performed by single-factor analysis, and the significant difference analysis was performed by t-test, P <0.05, P <0.01, P < 0.001.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. The method for obtaining cerebral cortex organoids of Alzheimer's disease is characterized by comprising the following steps:

s2, carrying out subculture on the iPSCs containing the double mutant genes, and removing feeder layer cells to obtain purified iPSCs;

the first culture medium is: LIF-free iPSCs serum-free medium;

the components of the composition are knock-out DMEM, KSR8 with the mass fraction of 20%, 100 × L-GlutaMax, 100 × NEAA, 1000 × 2-ME, and the concentration of the 2-ME is 10 mM;

the second culture medium is: a neuroepithelial differentiation medium;

the components are DMEM-F12, 100 × N2 supplement, 100 × L-GlutaMax, 100 × NEAA, MouseNgin;

the third culture medium is: a cerebral cortex differentiation culture medium,

the ingredients of the composition are DMEM/F12, Neurobasal medium, 50 × B27, 100 × N2 supplement, 100 × L-GlutaMax, 100 × NEAA, 100 × P/S and 2 mu g/L Mouse IGF-I.

2. The method of claim 1, wherein the purified iPSCs is provided at a concentration of 1 × 10S 3, wherein said purified iPSCs are administered to said patient in need of treatment of said disease⁴One by 100. mu.l.

3. The method of claim 1, wherein in step S3, cell culture plate a is a low-adhesion U-bottom 96-well plate and cell culture plate b is a low-adhesion 6-well plate.

4. The method of obtaining a cerebral cortical organoid of alzheimer' S disease of claim 1, wherein in S3, Rock inhibitor Y27362 is added to the first culture medium.

5. The method of obtaining a cerebral cortical organoid of alzheimer's disease of claim 4, wherein said Rock inhibitor Y27362 is provided at a final concentration of 50 μ M.

6. The method of claim 1, wherein in step S3, the translucent neuroepithelial bud-like projections extending from the periphery of the embryoid body are coated with Matrigel.

7. The method of claim 1, wherein the culture conditions in S3 are 37 ℃ and 5% volume CO₂And balancing humidity.

8. An alzheimer's disease cerebral cortical organoid obtainable by the method of any one of claims 1-7.

9. Use of the cerebral cortical organoids of alzheimer's disease of claim 8 as a model of alzheimer's disease.

10. The use of the cerebral cortical organoids of alzheimer's disease of claim 8 as a platform for screening of alzheimer's disease therapeutic drugs.