CN111635889B

CN111635889B - Compositions and methods for reprogramming human astrocytes into neurons or brain-like organs

Info

Publication number: CN111635889B
Application number: CN202010477310.8A
Authority: CN
Inventors: 邵志成; 方诗; 许锦洪
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2020-05-29
Filing date: 2020-05-29
Publication date: 2023-04-28
Anticipated expiration: 2040-05-29
Also published as: CN111635889A

Abstract

The invention belongs to the technical field of biology, and relates to a composition and a method for reprogramming human astrocytes into neurons or brain-like organs. A composition for efficiently reprogramming human astrocytes into neurons comprising a substance capable of achieving OCT4 overexpression, a substance that knocks down cell cycle regulatory factor p53, and a small molecule drug capable of inducing reprogramming human astrocytes into neurons selected from one or more of the following four small molecule drugs: CHIR99021, SB431542, repnox or Y27632. Compared with the prior art, the invention combines the regulation and control of two genes, obtains a brand-new small molecule drug combination which can effectively reprogram Cheng Ren astrocytes as neurons and brain-like organs through massive screening, and verifies the reliability of the screened system on astrocytes differentiated from human embryonic stem cells H9 and astrocytes of tissues beside glioma patients.

Description

Compositions and methods for reprogramming human astrocytes into neurons or brain-like organs

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a composition and a method for reprogramming human astrocytes into neurons or brain-like organs.

Background

In 2017, stem Cell Reports issued on-line a research paper "Direct Generation of Human Neuronal Cells from Adult Astrocytes by Small Molecules" of Pei steel research group of national academy of sciences biochemistry and cell biology, reporting the research results of using small molecule compound combinations to achieve direct conversion of adult astrocytes into neural cells.

Pei Gang yard group uses 6 small molecule drugs to reprogram human astrocytes into functional neurons, and fig. 1 is a schematic diagram. The specific scheme is as follows: human adult astrocytes were inoculated into polylysine-pretreated astrocyte growth medium (AM). After two days the cells reached 90% density and the growth medium was replaced with neurobasal+1% B27+1% N2+BDNF (20 ng/ml) +GDNF (20 ng/ml) +IGF (20 ng/ml) +Vc (0.2. Mu.M) +dibutyryl-cAMP (100. Mu.M) +laminin (1. Mu.g/ml) (called NM medium). On this basis, six small molecule drugs 0.5mM VPA (Calbiochem), 3. Mu.M Chir99021 (Selleck), 1. Mu.M Repsox (Biovision), 10. Mu.M Forskolin (Cayman), 2. Mu. M i-BET151 (MedChem Express), 10. Mu.M ISX-9 (MedChem Express) were added for neuronal induction. The induction medium containing the small molecule drug was changed every 4 days for 20 days for induction with the addition of human astrocytes to support neuronal maturation when necessary. This induction method allows MAP2 and neen positive neurons to be detected on day 12. The efficiency of inducing reprogramming of human astrocytes into neurons was 8% compared to the total amount of starting cells. There was no proliferation of cells during this induction.

Chinese patent CN111073855a discloses a method for inducing astrocytes to transdifferentiate into serotonergic neurons and application thereof, and the invention discloses a method for inducing astrocytes to directly reprogram in vitro by using different transcription factor combinations to generate functional serotonergic neurons, and transplanting the obtained neurons into brain to survive. The addition of small chemical molecules based on the use of transcription factors can further increase the efficiency and maturity of serotonergic neurons. The serotonergic neuron produced by the method can be used for nerve regeneration, simulation of various nervous system diseases and drug screening.

Although the technology of the Pei Gang subject group described above is also disclosed in chinese patent CN111073855A in which Cheng Ren astrocytes can be reprogrammed as neurons, there are some drawbacks in the application of this reprogramming method to regeneration of central nervous tissue, such as: (1) Astrocytes are reprogrammed directly to the point that the neurons do not proliferate in large amounts after mitosis, and therefore a large number of target neurons cannot be obtained; (2) Such reprogrammed cells do not have the ability to self-assemble into human brain tissue, such as brain-like organs and the like; (3) This reprogramming method is a 2D system with no 3D brain tissue structure and functional connections.

Disclosure of Invention

The present invention is directed to a composition and method for reprogramming human astrocytes into neurons or brain-like organs that overcomes the above-described drawbacks of the prior art.

The inventors of the present application found through a great deal of experimental verification and mechanism study that the drawbacks existing in the prior art as described above are caused by the following reasons:

1. certain signaling pathways or epigenetic modifications are only capable of reprogramming astrocytes to produce specific neuronal subtypes, with poor plasticity.

2. The neurons obtained by reprogramming are mostly mitotic late-stage neurons, and the neurons belonging to terminal differentiation do not have proliferation capacity; without passing through a neural precursor stage with proliferation capability; thus, a large amount of the target cells cannot be obtained.

3. The induction system is a 2D culture system and cannot form a 3D brain tissue structure.

The inventor of the application researches and discovers that the defects can be remedied by regulating the expression of the dryness-related genes and activating specific signal channels to induce a neural precursor stage of massive proliferation and combining a 3D induction culture technology to realize the construction of brain-like organs.

Therefore, the invention discovers that the combination of a pair of genes and four small molecule drugs can effectively reprogram Cheng Ren astrocytes into neurons through a large number of screening. In combination with 3D induction culture techniques, reprogrammed astrocytes can be cultured into 3D brain-like organs. By regulating the spinal cord development induction signal molecules, reprogrammed astrocytes can be induced into spinal cord organoids which are expected to be used in cell therapy for nerve tissue repair such as spinal cord injury and cerebral ischemia injury in the future.

The aim of the invention can be achieved by the following technical scheme:

in a first aspect of the invention, there is provided a composition that can efficiently reprogram human astrocytes into neurons: the compositions include agents that are capable of achieving OCT4 overexpression and agents that knock down cell cycle regulatory factor p 53.

The substance for realizing the over-expression of OCT4 can be a substance for realizing the over-expression of OCT4 commonly used in the field.

The substance for knocking down the cell cycle regulatory factor p53 may be any substance commonly used in the art for knocking down the cell cycle regulatory factor p 53.

In one embodiment of the first aspect of the invention, a substance is provided that simultaneously overexpresses OCT4 and knocks down p53, and the vector pCXLE-hOCT3/4-shp53-F, addgene accession #27077 is selected. pCXLE-hOCT3/4-shp53-F, addgene accession #27077 is known in the art.

In a second aspect of the invention, there is provided a composition capable of inducing reprogramming of human astrocytes into neurons, said composition being selected from one or more of the following four small molecule drugs: CHIR99021, SB431542, repnox or Y27632.

In one embodiment of the second aspect of the invention, preferably CHIR99021 is used in an amount of 1.5 μm, SB431542 is used in an amount of 5 μm, repox is used in an amount of 1 μm, and Y27632 is used in an amount of 10 μm.

In one embodiment of the second aspect of the invention, the composition capable of inducing reprogramming of human astrocytes into neurons is a combination of CHIR99021, SB431542, repnox and Y27632.

In one embodiment of the second aspect of the invention, preferably CHIR99021, SB431542, repbox and Y27632 are used in combination in an amount of 1.5 μm, SB431542 in an amount of 5 μm, repbox in an amount of 1 μm and Y27632 in an amount of 10 μm.

In a third aspect of the invention, there is provided a composition that can efficiently reprogram human astrocytes into neurons: the composition comprises a substance capable of realizing the overexpression of OCT4, a substance for knocking down a cell cycle regulating factor p53 and a small molecule drug capable of inducing the reprogramming of human astrocytes into neurons, wherein the small molecule drug capable of inducing the reprogramming of human astrocytes into neurons is selected from one or more of the following four small molecule drugs: CHIR99021, SB431542, repnox or Y27632.

In one embodiment of the third aspect of the invention, preferably a substance is provided that simultaneously overexpresses OCT4 and knocks down p53, the vector pCXLE-hctc 3/4-shp53-F, addgene number #27077 is selected.

In one embodiment of the third aspect of the invention, preferably CHIR99021 is used in an amount of 1.5 μm, SB431542 is used in an amount of 5 μm, repox is used in an amount of 1 μm, and Y27632 is used in an amount of 10 μm.

In one embodiment of the third aspect of the invention, the small molecule drug capable of inducing reprogramming of human astrocytes into neurons is a combination of CHIR99021, SB431542, repnox and Y27632.

In one embodiment of the third aspect of the present invention, preferably, CHIR99021, SB431542, repbox and Y27632 are used in combination, wherein CHIR99021 is used in an amount of 1.5 μm, SB431542 is used in an amount of 5 μm, repbox is used in an amount of 1 μm, and Y27632 is used in an amount of 10 μm.

In a fourth aspect of the invention, a method of reprogramming human astrocytes into neurons is provided.

Specifically, the method of reprogramming human astrocytes into neurons is as follows:

culturing human astrocytes to a density of 80-90% with AM medium;

adding a substance capable of realizing OCT4 overexpression and a substance for knocking down a cell cycle regulating factor p53 into human astrocytes to realize OCT4 overexpression and p53 knocking down;

culturing in AM medium for 2 days, and adding small molecule medicine capable of inducing human astrocyte to reprogram into neuron for induction from the third day;

starting with the addition of a small molecular drug capable of inducing the reprogramming of human astrocytes into neurons, the culture medium is changed into a neuron culture medium, the culture medium is changed for 2 or 3 days, the small molecular drug capable of inducing the reprogramming of human astrocytes into neurons is not added into the neuron culture medium any more on the 8 th day, and the fresh neuron culture medium is changed for 2 to 3 days and is continuously cultured for 21 days.

The above process achieves the reprogramming of human astrocytes into neurons.

In one embodiment of the fourth aspect of the invention, the neuronal medium is preferably selected as Neurobasal medium+Glutmax (1:100) +NEAA (1:100) +B27 supplements (1:50) +BDNF (10 ng/ml) +GDNF (10 ng/ml) +L-Ascorbic acid 2-phosphate (200 uM) + Penicillin andStreptomin (1:200).

In one embodiment of the fourth aspect of the invention, preferably, after the end of the culture, immunostaining identification is performed with the neuronal marker MAP2 and the proportion of positive cells is counted.

In one embodiment of the fourth aspect of the invention, preferably a substance is provided that simultaneously overexpresses OCT4 and knocks down p53, the vector pCXLE-hOCT3/4-shp53-F, addgene number #27077 is selected.

In one embodiment of the fourth aspect of the invention, preferably CHIR99021 is used in an amount of 1.5 μm, SB431542 is used in an amount of 5 μm, repox is used in an amount of 1 μm, and Y27632 is used in an amount of 10 μm.

In one embodiment of the fourth aspect of the invention, the small molecule drug capable of inducing reprogramming of human astrocytes into neurons is a combination of CHIR99021, SB431542, repnox and Y27632.

In one embodiment of the fourth aspect of the invention, preferably, CHIR99021, SB431542, repbox are used in combination with Y27632 in an amount of 1.5 μm, SB431542 in an amount of 5 μm, repbox in an amount of 1 μm, and Y27632 in an amount of 10 μm.

In a fifth aspect of the invention, there is provided a method of inducing human astrocytes into 3D cortical organoids (cortex organoids).

Specifically, the method of inducing human astrocytes into 3D cortical organoids (cortex organoids) is as follows:

culturing human astrocytes to a density of 80-90% with AM medium;

starting from adding small molecular drugs capable of inducing the reprogramming of human astrocytes into neurons, changing the culture medium into a neuron culture medium, changing the culture medium for 2 or 3 days, and on the 8 th day, not adding small molecular drugs capable of inducing the reprogramming of human astrocytes into neurons into the neuron culture medium, and keeping the fresh neuron culture medium to be changed for 2 to 3 days for continuous culture;

a population of cells producing a large rosette-like (rosette) structure was induced on day 14, these cells were transferred to a 3D culture system, and the induced cells aggregated into a spherical structure;

by day 21, spheroids with neuroepithelial-like budding growth were embedded with Matrigel matrix, and allowed to grow to a size of 3-4mm for 85-95 days, forming brain organoids similar to those induced by human pluripotent stem cells.

By modulating spinal cord development induction signaling molecules, reprogrammed astrocytes were successfully induced into spinal cord organoids (ADSC-organons), preferably in the following manner:

on days 16-18, bFGF (20 ng/ml) and retinoic acid (100 nM) were added and cells were induced to aggregate into spheres in neuronal medium. Beginning on day 19, the SHH signaling agonist SAG (500 nM) was added to the nerve medium and after 10 days of SAG induction, BMP4 (15 ng/ml) was added for 3 days.

While cortical organoids do not require bFGF (20 ng/ml) and retinoic acid (100 nM), as well as SAG, BMP4 treatment.

In one embodiment of the fifth aspect of the invention, preferably, upon completion of BMP4 induction, the culture is continued on a shaker by embedding with matrix gel Matrigel and then transferring to an ultra-low adsorption petri dish.

Compared with the prior art, the invention combines the regulation and control of two genes, obtains a brand-new small molecular medicine combination which can effectively reprogram Cheng Ren astrocytes as neurons through massive screening, and verifies the reliability of the screened system on astrocytes differentiated from human embryonic stem cells H9 and astrocytes of tumor-side tissues of glioma patients.

The screened medicine combination CSBRY can induce and accelerate the reprogramming of the primary astrocytes of human beings and has strong capability of promoting the proliferation of cells. Human astrocytes, induced by CSBRY, re-enter the neurogenesis process. After transfer to the 3D culture system, the induced cells aggregate into organoids (organoids). By immunostaining identification, the neural progenitor cell marker SOX2 was observed ⁺ And forebrain specific progenitor cell marker PAX6 ⁺ Is demonstrated to be capable of culturing into 3D cortical organoids (AD-organoids). These characteristics are not available in other existing astrocyte reprogramming methods.

Meanwhile, by regulating the spinal cord development induction signal molecule, reprogrammed astrocytes were successfully induced into spinal cord organoids (ADSC-organons), which was also the first report that human astrocytes could be reprogrammed into spinal cord organoids.

Drawings

FIG. 1 is a diagram of a prior art technique for reprogramming human astrocytes into functional neurons using 6 small molecule drugs.

Fig. 2 includes fig. 2A, 2B, 2C;

fig. 2A: the induction process schematic diagram for realizing reprogramming of human astrocytes into neurons is realized in the invention;

fig. 2B: representative images of immunostaining with neuronal marker MAP2 at day 21 of culture;

fig. 2C: statistics of proportion of immunostained MAP2 positive cells for control and 15 small molecule drug alone treatment groups.

Fig. 3 includes fig. 3A, 3B, 3C, 3D, 3E, 3F;

fig. 3A: representative figures of immunostaining with neuronal marker MAP2 for control and different combination treatment groups of CHIR99021, SB431542, repnox and Y27632;

fig. 3B: immunostaining MAP2 positive cell proportion statistical plots were performed for the control group and different combination treatment groups of CHIR99021, SB431542, repbox and Y27632, and the data showed mean ± standard deviation;

fig. 3C: astrocytes derived from directional differentiation of H9 embryonic stem cells were reprogrammed to an immunostaining pattern of MAP2 positive neurons under CSBRY treatment;

fig. 3D: carrying out immunostaining MAP2 positive cell proportion statistical diagram on astrocytes from H9 embryonic stem cell directional differentiation under CSBRY treatment, wherein the data show average value +/-standard deviation;

fig. 3E: normal astrocytes from glioma patient were reprogrammed under CSBRY treatment to MAP2 positive neuronal immunostaining patterns;

fig. 3F: MAP2 positive cell proportion statistics for immunostaining of normal astrocytes from glioma patients under CSBRY treatment, data showing mean ± standard deviation.

FIG. 4 includes FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F, FIG. 4G, FIG. 4H

Fig. 4A: the invention realizes the induction process schematic diagram of the human astrocyte cultured into the 3D cortex organoid;

fig. 4B: exemplary figures at

day

8, 14, 42, 90 in bright field during 3D cortical organoid culture, scale is the figure at day 8: 100uM; graph at day 14: 400uM; graph at day 42: 1000uM; graph at day 90: 1000uM;

fig. 4C: culture to week 7, section staining showed formation of neural progenitor cell marker SOX2 ⁺ And forebrain specific progenitor cell marker PAX6 ⁺ Ventricular zone-like (VZ) structure and TUJ1 ⁺ /MAP2 ⁺ The neuron skin layer structure, the scale is 25uM;

fig. 4D: culturing to week 10, and staining the sections to show SOX2 ⁺ And PAX6 ⁺ The VZ structural region is continuously increased, and scales are respectively 50uM,25uM and 50uM;

fig. 4E: the astrocytes which independently regulate and control the expression of OCT4 and p53 cannot realize rapid proliferation of cells, and when the astrocytes are added with small molecule medicine combination CSBRY for treatment, the number of the cells rapidly proliferates, and the proliferation of the cells is TUJ1 at most ⁺ /MAP2 ⁺ NeuronsData show mean ± standard deviation;

fig. 4F: the figure shows that astrocytes which independently regulate and control the expression of OCT4 and p53 cannot form cortical Organoids (called AD-Organoids), and the cortical Organoids can be formed by adding small molecule drug combination CSBRY treatment on the basis, and the scale is 1000uM;

fig. 4G: astrocytes treated by regulating OCT4 and p53 expression and small molecule drug combination CSBRY realize rapid proliferation of cells, and most of the proliferated cells are TUJ1 ⁺ /MAP2 ⁺ Neurons, scale 25uM;

fig. 4H: MAP2 positive cells and TUJ1 positive cell proportion statistics of immune staining of astrocytes treated by regulating OCT4 and p53 expression and small molecule drug combination CSBRY; the data show mean ± standard deviation.

FIG. 5 includes FIGS. 5A, 5B, 5C

Fig. 5A: representative figures of immunostaining identification of human astrocyte HA1800 with different markers;

fig. 5B: the human astrocyte HA1800 was identified by immunostaining and statistics show the percentage of each marker.

Fig. 5C: human astrocytes were seeded onto low-adherent 24-well plates, 50 cells per well, and cultured with a neurosphere culture system containing bFGF and EGF. No neurospheres were found after 14 days, but astrocytes were induced by Op53-CSBRY to produce neurospheres; the scale is 200uM.

FIG. 6 includes FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, FIG. 6G, FIG. 6H, FIG. 6I, FIG. 6J, FIG. 6K, FIG. 6L, FIG. 6M, FIG. 6O

Fig. 6A: the invention realizes the induction process schematic diagram of the 3D spinal cord organoid cultured by human astrocytes;

fig. 6B, C: figures at

fields

4 and 7 Zhou Zaiming in the 3D spinal organoid culture process, scale 1000uM;

fig. 6D: schematic representation of progenitor cell zone expression patterns during spinal cord development;

fig. 6E, F, G: culturing until week 7, section staining showed high expression of PAX6, ventral precursor marker NKX6.1, dorsal precursor marker OLIG3 in 3D spinal cord organoids (hADSC-organons), scale 25uM;

fig. 6H: culture to week 7, section staining showed NKX6.1 ⁺ Cells form ventricular partition-like structures, dorsal marker OLIG3 ⁺ Cells located on ventral NKX6.1 ⁺ Outside the cell, showing a dorsal-ventral-like spatial distribution, scale 25uM;

fig. 6I: culturing to week 7, and section staining showed ventral motor neuron precursor cells OLIG2 ⁺ Mainly with ventral NKX6.1 ⁺ Co-locating cells to form an ventral structure, with a scale of 25uM;

fig. 6J, K, L, M: incubation to week 10, section staining indicated detection of CHAT ⁺ 、HB9 ⁺ Motor neuron of GABA ⁺ Inhibitory neurons and VGLUT1 ⁺ Is 25uM on the scale of excitatory neurons;

fig. 6N, O: transcriptome analysis of AD-Organ and hADSC-Organ induced by human astrocytes for 7 weeks showed that induced hADSC-Organ widely expressed genes specifically expressed by spinal cord at the developmental stage.

Detailed Description

In a second aspect of the invention, there is provided a composition capable of inducing reprogramming of human astrocytes into neurons, said composition being selected from one or more of the following small molecule drugs: CHIR99021 (1.5 μm), VPA (5 mM), Y27632 (10 μm), SB431542 (5 μm), XAV939 (10 μm), forskolin (10 μm), JQ1 (50 nM), PD0325901 (10 μm), repbox (1 μm), ISX9 (10 μm), LDN193189 (100 nM), TTNPB (500 nM), SAG (100 nM), SU5402 (10 μm) and DAPT (2.5 μm).

In one embodiment of the second aspect of the present invention, preferably, the composition is selected from one or several of the following four small molecule drugs: CHIR99021, SB431542, repnox or Y27632.

culturing human astrocytes to a density of 80-90% with AM medium;

The above process achieves the reprogramming of human astrocytes into neurons.

In one embodiment of the fourth aspect of the invention, the neuronal medium is preferably selected as Neurobasal medium+Glutmax (1:100) +NEAA (1:100) +B27 supplements (1:50) +BDNF (10 ng/ml) +GDNF (10 ng/ml) +L-Ascorbic acid 2-phosphate (200 uM) + Penicillin and Streptomin (1:200).

culturing human astrocytes to a density of 80-90% with AM medium;

The invention will now be described in detail with reference to the drawings and specific examples.

Example 1

The invention firstly screens out the combination of a pair of genes and four small molecule drugs, and can efficiently reprogram human astrocytes into neurons.

In order to obtain a large number of neuronal cells and 3D brain-like organs, the over-expression of the key cell reprogramming factor OCT4 is considered to promote the reprogramming process of human astrocytes in the research process of the application, and the knocking down of the cell cycle regulating factor p53 can promote the proliferation of intermediate reprogramming cells.

By simultaneously overexpressing OCT4 and knockdown p53 in human primary astrocytes, (this application will refer to as "Op53" in the following description), although in the present application studies OCT4 and knockdown p53 have been effectively overexpressed in astrocytes, it was found that only a few cells could be reprogrammed to MAP2 ⁺ And (3) cells.

To increase the conversion, 15 small molecule drugs involved in neural differentiation and somatic reprogramming were selected in the present application. The 15 small molecules initially screened were: CHIR99021 (1.5 μm), VPA (5 mM), Y27632 (10 μm), SB431542 (5 μm), XAV939 (10 μm), forskolin (10 μm), JQ1 (50 nM), PD0325901 (10 μm), repbox (1 μm), ISX9 (10 μm), LDN193189 (100 nM), TTNPB (500 nM), SAG (100 nM), SU5402 (10 μm) and DAPT (2.5 μm).

The induction process is shown in fig. 2A: human astrocytes were cultured with AM medium (e.g., selected scientific, HA1800, cat# 1801) at about 80-90% density, and vectors (pCXLE-hct 3/4-shp53-F, addgene # 27077) with simultaneous OCT4 overexpression and p53 knockdown were transferred to astrocytes using the LONZA electrotransfer kit, and cultured with AM medium until day 3, followed by small molecule induction, with a 2 or 3 day change of medium. From the start of administration, the medium was changed to Neurobasal medium (Thermo Fisher Scientific) +Glutmax (1:100,Thermo Fisher Scientific) +NEAA (1:100,Thermo Fisher Scientific) +B27 supplements (1:50,Thermo Fisher Scientific) +BDNF (10 ng/ml, peprotech) +GDNF (10 ng/ml, peprotech) +L-Ascorbic acid 2-phosphate (200 uM, sigma) + Penicillin and Streptomin (1:200,Thermo Fisher Scientific) (referred to as neuronal medium). On day 8, the small molecule drug was removed, the fresh neuronal medium was kept for 2-3 days and continued to be cultured for 21 days, immunostaining identification was performed with neuronal marker MAP2 and the positive cell proportion was counted, and the results are shown in FIGS. 2B and 2C.

Studies have found that CHIR99021, SB431542, repox and Y27632 can significantly promote reprogramming of human astrocytes into neurons.

Positive cells obtained by single treatment of CHIR99021, SB431542, repSox and Y27632 all had a proportion of higher than 8%.

To further increase reprogramming efficiency, the present application tested different combinations of these four small molecules.

CHIR99021 is abbreviated as C, SB431542 is abbreviated as SB, repbox is abbreviated as R, Y-27632 is abbreviated as Y, the combination of CHIR99021 and SB431542 is abbreviated as CSB, the combination of CHIR99021, SB431542, repbox and Y-27632 is abbreviated as CSBRY, and the like, and other combinations are similarly named.

The combination of CHIR99021, SB431542, repox and Y-27632, i.e. CSBRY, was found to significantly increase the conversion efficiency of astrocytes (fig. 3a, b). Studies have also found that CSBRY can also significantly promote the efficiency of H9 embryonic stem cell-directed differentiation-derived astrocytes to reprogram into MAP2 neurons (fig. 3c, d). To verify the results of the above experiments, the present application again obtained and cultured normal astrocytes derived from adult glioma patients. Whereas 2/3 of patient astrocytes (more than 80% of all cells were GFAP positive) samples could be reprogrammed to neurons, but with lower efficiency than human primary astrocytes (fig. 3e, f). Thus, these results above demonstrate that Op53 in combination with CSBRY can efficiently reprogram human astrocytes into neurons in vitro.

In conjunction with 3D induction techniques, reprogrammed astrocytes can be cultured into 3D cortical organoids (cortex organs).

The present invention observed that Op53-CSBRY induced a large population of cells that generated rosette-like (rosette) structures on day 14. This suggests that it is possible that primary astrocytes were induced into the neurogenic process by Op 53-CSBRY. To confirm this judgment, on day 14, these cells were transferred to a 3D culture system, and the induced cells aggregated into a spherical structure. Around day 21, spheroids with neuroepithelial-like sprouting growth were embedded with Matrigel, and around day 90 could grow to a size of about 3-4mm, similar to brain organoids induced by human pluripotent stem cells (fig. 4a, b). At week 7, the neuro progenitor marker SOX2 was observed ⁺ And forebrain specific progenitor cell marker PAX6 ⁺ Ventricular zone-like (VZ) structure and TUJ1 ⁺ /MAP2 ⁺ Neuronal cortex structures (fig. 4C). SOX2 ⁺ And PAX6 ⁺ The VZ layer increased continuously for 10 weeks (fig. 4D). To determine whether an astrocyte-derived organ includes multiple cortex layers, the deep cortex neuronal marker CTIP2 and the upper cortex neuronal marker SATB2 were stained. CTIP2 was found in the organoids at 10 weeks ⁺ And SATB2 ⁺ Cells showed stratified expression. This astrocyte reprogramming induction-derived cortical organoid is defined herein as AD-Organoids. It was also found in the present application that astrocytes, which regulate OCT4 and p53 expression alone, are unable to form AD-Organoids. The cells are rapidly proliferated by the treatment of small molecule drug combination CSBRY, and the proliferated cells are TUJ1 ⁺ /MAP2 ⁺ Neurons (fig. 4e, g, h). This suggests that CSBRY combinations may induce and accelerate neurogenesis in the conversion of human astrocytes to neurons. To confirm the origin of AD-organics, the present application found that more than 80% of cultured human astrocytes were GFAP and S100B positive with few MAP2 positive neurons. However, some SOX2 was found in human astrocyte cultures ⁺ And PAX6 ⁺ Is described (FIGS. 5A, B). To examine whether these progenitor cells are neural stem cells, the present application inoculates low-adherence 24-well plates with 50 astrocytes per well, and cultures with neurosphere culture system containing bFGF and EGF. After 14 days, no neurospheres were found in the untreated 8 culture wells. Astrocytes, however, were induced by Op53-CSBRY to produce neurospheres (fig. 5C). These results indicate that AD-Organoids are produced by reprogramming primary astrocytes, rather than by contamination of Neural Stem Cells (NSCs).

The present application studies induced astrocytes as spinal cord tissue organoids by modulating FGF, SHH and BMP signaling pathways.

The present application devised a spinal cord organoid of human astrocyte origin (abbreviated as hADSC-Organ) induction protocol:

when a large number of neural precursor cells were present on day 14, the cells were transferred to an ultra low adhesion 24-well plate (Corning). On the 16 th day to the 18 th day,bFGF (20 ng/ml, peprotech) and retinoic acid (100 nM, sigma) were added and cells were induced to aggregate into spheres in neuronal medium. Beginning on day 19, the SHH signaling pathway agonist SAG (500nM,EMD Millipore) was added to the nerve medium, and after 10 days of SAG induction BMP4 (15 ng/ml, peprotech) was added for 3 days. Upon completion of BMP4 induction, spheres in 24 well plates were embedded in Matrigel gel and then transferred to ultra-low adsorption 60mm dishes and placed on a shaker for further culture (fig. 6A). After 7 weeks of induction, hADSC-Organ grew to a size of 2-3mm (FIGS. 6B, C), and different dorsal and ventral progenitor markers were analyzed. PAX6, ventral precursor marker NKX6.1, dorsal precursor marker OLIG3 was found to be highly expressed in hASCO-Organ (FIG. 6E-G), NKX6.1 ⁺ The cells form a chamber partition like structure. Importantly, the backside marker OLIG3 ⁺ Cells located on ventral NKX6.1 ⁺ Outside the cell, a spatial distribution resembling that of the dorsal-ventral side is shown (fig. 6H). Ventral motor neuron precursor cell OLIG2 ⁺ Mainly with ventral NKX6.1 ⁺ Cells co-localized, forming ventral structures (fig. 6I). These results indicate that the directional induction protocol of the present application successfully induced ventral and dorsal side formation of hADSC-Organ. At the same time, the present application explores whether hADSC-Organ can produce different subtypes of spinal cord neurons, such as GABAergic neurons, glutaminergic neurons, or cholinergic spinal motor neurons. As shown in FIG. 6J-K results, CHAT was detected by hADSC-Organ for 10 weeks ⁺ 、HB9 ⁺ Is a motor neuron, GABA neuron and VGLUT1 positive excitatory neuron. To further demonstrate the spinal cord identity of ADSC-Organ, astrocyte-induced AD-Organoids and ADSC-Organ were also subjected to gene expression profiling analysis, which revealed that ADSC-Organ highly expressed spinal cord-specific expressed genes such as MNX1, ISL1, CHAT, NKX6-1, NKX2-2, and a plurality of HOX family genes (FIG. 6N, O). Thus, it was found from the above studies that under specific conditions in vitro, human astrocytes were reprogrammed to produce human dorsal-ventral progenitor cells and neurons of the main subtype, and had spinal cord tissue structures and gene expression patterns similar to those of the human development stage, confirming the organoids of the spinal cord.

The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.

Claims

1. A method of reprogramming human astrocytes into neurons or brain-like organs using the composition: wherein the composition comprises a substance capable of realizing the over-expression of OCT4, a substance for knocking down a cell cycle regulating factor p53 and a small molecule drug for promoting the induction of the reprogramming of human astrocytes into neurons or brain-like organs, wherein the small molecule drug for promoting the induction of the reprogramming of the human astrocytes into the neurons or brain-like organs is a combination of CHIR99021, SB431542, repSox and Y27632, the dosage of CHIR99021 is 1.5 mu M, the dosage of SB431542 is 5 mu M, the dosage of RepSox is 1 mu M, and the dosage of Y27632 is 10 mu M;

the composition is used to synergistically reprogram human astrocytes into neurons and induce the production of 3D cortical and spinal organoids.

2. The method according to claim 1, comprising the steps of:

culturing human astrocytes to a density of 80-90% with AM medium;

culturing in AM medium for 2 days, and adding small molecule medicine for promoting induction of human astrocyte reprogramming into neuron or brain-like organ for induction from the third day;

starting from adding small molecular drugs for promoting the induction of the reprogramming of the human astrocytes into neurons or brain-like organs, changing the culture medium into a neuron culture medium, changing the culture medium for 2 or 3 days, and on the 8 th day, no small molecular drugs for promoting the induction of the reprogramming of the human astrocytes into neurons or brain-like organs are added into the neuron culture medium, and keeping the fresh neuron culture medium for 2 to 3 days for continuous culture until 21 days.

3. The method according to claim 1, comprising the steps of:

culturing human astrocytes to a density of 80-90% with AM medium;

starting from adding small molecular drugs for promoting the induction of the reprogramming of the human astrocytes into neurons or brain-like organs, changing the culture medium into a neuron culture medium, changing the culture medium for 2 or 3 days, and on the 8 th day, not adding small molecular drugs for promoting the induction of the reprogramming of the human astrocytes into neurons or brain-like organs into the neuron culture medium, and keeping the fresh neuron culture medium for 2 to 3 days for continuous culture;

inducing a large number of cell populations with rosette-like structures on day 14, transferring the cells into a 3D culture system, and aggregating the induced cells into spheroids;

by day 21, spheroids were sprouted with neuroepithelial patterns and grown to a size of 3-4mm by day 21 with Matrigel embedding, and were able to grow to a size of 85-95 days, forming a cortical organoid similar to that induced by human pluripotent stem cells.

4. The method of claim 3, wherein the reprogrammed astrocytes are successfully induced into spinal cord organoids by modulating spinal cord development induction signaling molecules, and wherein for spinal cord organoid directed preparations, at days 16-18, bFGF 20ng/ml and retinoic acid 100nM are added for 3 days, cells are induced to aggregate into spheres in neuronal medium, beginning at day 19, SHH signaling pathway agonist SAG 500nM is added to neuronal medium, and after 10 days of SAG induction BMP4 ng/ml is added for 3 days.

5. The method of claim 3, wherein upon completion of BMP4 induction, the culture is continued on a shaker by embedding with matrix gel Matrigel and transferring to an ultra-low adsorption petri dish.