WO2023143637A1 - Construction and use of practical brain-like microorgan - Google Patents

Abstract

The present invention provides the construction and use of a practical brain-like microorgan. Disclosed is an optimized method for preparing a brain-like microorgan. The method comprises: inducing and culturing the brain-like microorgan; and at an appropriate culture stage, harvesting the early brain-like microorgan, which can continue to grow and differentiate in vivo to generate a mature and functional brain-like microorgan.

Description

Construction and application of a practical brain micro-organ

This application claims the priority of the application number CN202210092811.3 and the title of the invention "Construction and application of a practical brain micro-organ" submitted on January 26, 2022, the entire content of which is incorporated herein by reference .

technical field

The invention belongs to the fields of neuroscience and regenerative medicine; more specifically, the invention relates to the construction and application of a practical brain-like micro-organ.

Background technique

With the increase of aging population and increasing social pressure, brain-related diseases have become an important class of diseases that endanger the quality of human life. The human brain is a highly ordered structure composed of various cell types, including neurons, glial cells, and vascular cells, among others. Modeling human brain development is extremely challenging due to the complex interplay of different signals in space and time.

Organoids are stem cell-derived 3D cultures that mimic the cellular and structural features of the human brain to some extent, making them an important source of transplant material for cell therapy. Previous studies have shown that human brain organoids can exhibit neuronal maturation and corresponding function when transplanted into the adult mouse cortex, while developing host-mediated vascular networks. These findings suggest that the use of brain organoid transplantation to treat neurological diseases holds great promise.

Previous studies have evaluated human cerebral organoids (human Cerebral Organoids, hCOs) by transplanting them into the rodent cortex to assess their integration and therapeutic effects, but after transplantation of such brain organoids, there are still a series of challenges, including: Transplantation in vivo Apoptosis occurs later, the survival rate is not ideal, and the in vivo differentiation ability (such as but not limited to the ability to differentiate into neuronal cells, pericyte-like and mature choroid plexus cells, astrocytes, pericyte-like cells or choroid plexus cells) is still insufficient. need to be strengthened.

In addition, mature brain organoids often form larger cell aggregates (up to 3-5 mm in diameter), necessitating the use of thicker catheters for organoid injection, which imposes a risk on healthy tissue throughout the procedure. More damage, a problem that is more pronounced when organoids are transplanted into deep brain regions.

Therefore, there is an urgent need in the field for a new brain organoid construction technology to minimize the impact on the host Brain damage.

Contents of the invention

The purpose of the present invention is to provide the construction and application of a practical brain micro-organ.

In the first aspect of the present invention, there is provided a method for preparing brain micro-organs, the method comprising: (1) cultivating pluripotent stem cells to obtain embryoid bodies (EBs); (2) cultivating the embryoid bodies of (1) body, induced by neuroectodermal differentiation conditions, to obtain cultures containing early brain micro-organs (groups) of suitable size, the diameter of the early brain micro-organs is 150-600 μm; preferably 200-500 μm; and (3 ) Isolation of early brain-like micro-organs (populations) from cultures of (2).

In one or more embodiments, the methods of step (1) and step (2) are in vitro methods.

In one or more embodiments, the early brain micro-organs include early brain micro-organ populations.

In one or more embodiments, after the step (2), step (3) is performed to isolate early brain micro-organs for transplantation in vivo.

In one or more embodiments, the pluripotent stem cells are selected from (but not limited to): pluripotent stem cells (hPSC), embryonic stem cells (ES) or induced pluripotent stem cells (iPS).

In one or more embodiments, the pluripotent stem cells are human or non-human mammalian embryonic stem cells.

In one or more embodiments, the non-human mammals include (but are not limited to): rodents (including mice, rats, hamsters, etc.), non-human primates (such as monkeys, orangutans, etc. ), livestock (such as cattle, sheep, dogs, pigs, rabbits, etc.).

In one or more embodiments, in step (3), the early brain micro-organoid (population) obtained in step (2) is separated from the culture medium, and added to a pharmaceutically/physiologically acceptable carrier.

In one or more embodiments, in step (3), the early brain micro-organs (groups) obtained in step (2) are separated from the culture medium, digested into isolated cells, and then added to the pharmaceutical/physiological in an acceptable carrier.

In one or more embodiments, the pharmaceutically/physiologically acceptable carrier includes a solvent, a suspending agent; for example, a buffer solution (pH7.2-7.6, preferably pH7.3-7.5, more preferably pH7.35- 7.45), physiological saline, etc.

In one or more embodiments, in step (1), the culture time is 5±1 days; preferably 5±0.5 days.

In one or more embodiments, in step (2), the culture time is 24-72 hours; preferably 36-60 hours; more preferably 40-55 hours; most preferably 48±2 hours.

In one or more embodiments, in step (2), the culture time is, for example, 38, 40, 42, 44, 46, 48, 50, 52, 54 hours, preferably 48 hours.

In one or more embodiments, in step (1), the medium is an embryoid body (induction) medium; preferably it is EB formation medium EB formation medium (such as purchased from Stem Cell Technologies).

In one or more embodiments, in step (2), the medium is organoid induction medium; preferably, it is Induction medium induction differentiation medium (such as purchased from Stem Cell Technologies).

In one or more embodiments, cultures are performed using low adsorption culture vessels.

In one or more embodiments, the culture container is a culture well plate, such as a 12, 24, 36, 48, 96, 192, or 384 well plate.

In one or more embodiments, in the step (1), with 9000±5000 (such as 9000±4000; preferably 9000±3000; preferably 9000±2000; more preferably 9000 ±1000) The density of cells per well was added to a low-adsorption 96-well U-bottom plate for culture.

In one or more embodiments, in the step (2), the embryoid body of (1) is transferred to a low-adsorption 24-well plate for culture.

In one or more embodiments, in (1), the pluripotent stem cells are pluripotent stem cells carrying a reporter gene or a tracer marker; preferably, the reporter gene is passed through a virus (such as lentivirus, adenovirus, virus, adeno-associated virus) transfection is introduced into the pluripotent stem cells; more preferably, the virus transfection is carried out by single cell suspension transfection method (short time single cell suspension transfection method).

In one or more embodiments, in (2), the step of matrigel coating is not included.

In another aspect of the present invention, an early brain micro-organ (group) is provided, which is prepared by any of the aforementioned methods.

In one or more embodiments, the early brain-like micro-organs (groups) form mature brain-like micro-organs after being transplanted into the brain, preferably (but not limited to) the striatum, hippocampus, and cortex. Organs which exhibit the following properties:

Contains structures surrounded by pericyte-like and mature choroid plexus cells (ChP epithelioid cells) (preferably significantly more than that contained in micro-organs cultured in vitro at the same time);

Neuronal differentiation occurs; preferably, it comprises neuronal cells expressing the mature neuronal marker MAP2;

Astrocyte differentiation occurs; preferably, it comprises neuronal cells expressing the astrocyte marker GFAP;

Pericyte-like differentiation occurs; preferably, it comprises PDGFRβ-positive pericytes (there are no or few such cells in micro-organs cultured in vitro at the same period);

Differentiation of choroid plexus cells occurs; preferably, it comprises TTR-positive choroid plexus cells (there are no or few such cells in micro-organs cultured in vitro at the same time); and/or

Cell stress and/or low level of apoptosis (good viability); preferably its caspase 3 staining level is significantly low (significantly lower than its caspase 3 level in micro-organs cultured in vitro at the same period).

In one or more embodiments, the early brain micro-organ (group) is transplanted by stereotaxic injection.

In one or more embodiments, the "low/lower" or "high/higher" refers to a statistically significant or significant "lower/lower" or "higher/higher" , such as "lower/lower" or "higher/higher" by 1% to 99% (such as 2%, 5%, 10%, 15%, 20%, 30%, 50%, 60% compared with the control , 80%, 90%, 95% or 98%).

In one or more embodiments, the control includes (but not limited to): a non-transplanted negative control, or an in vitro micro-organ; preferably, the in-vitro micro-organ is a micro-organ cultured at the same period in vitro.

In another aspect of the present invention, there is provided the application of any one of the aforementioned early brain micro-organoids (groups) for preparing a composition for brain transplantation, and the composition for brain transplantation is used to form a mature brain organoid ,thereby:

Increased structures surrounded by pericyte-like and mature choroid plexus cells (ChP epithelioid cells) in the brain;

Increase neuronal cells in the brain (such as neuronal cells expressing mature neuronal marker MAP2);

Increased astrocytes in the brain (such as neurons expressing astrocyte marker GFAP);

Increased pericytes in the brain (eg, PDGFRβ-positive pericytes); and/or

Increased choroid plexus cells in the brain (such as TTR-positive choroid plexus cells).

In another aspect of the present invention, the application of any one of the aforementioned early brain micro-organoids (groups) is provided for preparing a composition for brain transplantation, and the composition for brain transplantation is used for alleviating or treating brain damage.

In one or more embodiments, the brain injury includes (but not limited to): nerve injury, Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies, Huntington's disease, and amyotrophic lateral sclerosis.

In another aspect of the present invention, a composition for brain transplantation is provided, which includes: any one of the aforementioned early brain micro-organs (groups); and a pharmaceutically acceptable carrier.

In one or more embodiments, the amount of the early brain micro-organoid (population) in the composition for brain transplantation is an effective amount.

In one or more embodiments, the composition comprises a pharmaceutical composition.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1. In vivo construction of IVD brain organoids.

(A) Procedure for constructing IVD brain organoids.

(B) In vivo structure of IVD brain organoids.

(C) DAPI and STEM121 (human cell marker) staining of IVD brain organoids and equal amounts of isolated cells.

(D) MAP2 staining in IVD brain organoids.

(E) GFAP staining in IVD brain organoids.

Figure 2. Cellular organization and state analysis of IVD brain organoids.

(A) PDGFRβ staining in IVD organoids and hCOs.

(B) TTR staining in IVD organoids and hCOs.

(C) Staining of cleaved caspase3 in IVD organoids and hCOs.

Detailed ways

The inventors of the present invention have devoted themselves to the cultivation optimization and transplantation research of practical brain-like micro-organs. After in-depth research, they have revealed an optimized method for preparing brain-like micro-organs. The method includes inducing and culturing brain-like organoids. Early brain micro-organoids are harvested, which can continue to grow and differentiate in vivo, yielding mature, functional brain organoids.

the term

As used herein, "brain micro-organoid (population)" refers to a multicellular/microorganoid organoid of appropriate size obtained from pluripotent stem cells through the induced differentiation of the present invention, suitable for transplantation, and capable of producing a mature brain organoid in vivo. Tissues may also be referred to as grafts in the present invention.

As used herein, "organ" refers to a non-unicellular state comprising two or more adjacent tissue layers, wherein the tissue layers maintain some form of cell-cell and/or cell-matrix interactions to produce microscopic Architecture. In the present invention, unless otherwise specified, the organ is a brain organ.

As used herein, "micro-organ culture" or "micro-organ population" refers to an isolated population of cells, such as a graft having the microarchitecture of the organ or tissue from which the cells were isolated. That is, separated cells together form 3D structures that mimic/preserve spatial interactions, such as cell-cell, cell-matrix, and cell-matrix interactions.

As used herein, "isolated" means, for example, that "early brain micro-organoid(s)" have been independently distinguished from the culture system/induction system.

As used herein, the "mammal" is an animal of the class Mammalia of the subphylum Vertebrata of the phylum Chordata. The mammals include but not limited to primates (including humans), artiodactyls, carnivores, rodents and the like. Mammals in the present invention include humans and non-human mammals. The non-human mammals include, for example but not limited to, rodents (specifically such as mice, rats), primates (specifically such as apes, monkeys, orangutans), livestock (specifically such as rabbits, dogs, rabbits, pigs) , cattle, sheep, horses) and so on.

In some embodiments of the present invention, mice are used as model organisms. Compared with humans, it is very close to humans in terms of genome composition, individual development, metabolic mode, organ anatomy, disease pathogenesis, etc.; Some conditions applicable to mice listed in the invention can be applied to other mammals such as humans without any doubt.

As used herein, the words "comprising", "having" or "comprising" include "comprising", "consisting essentially of", "consisting essentially of", and "consisting of";" "Mainly consist of", "essentially consist of" and "consist of" belong to the sub-concepts of "contain", "have" or "include".

As used herein, "functional" means that the spermatozoa obtained according to the described methods have the same or similar functions as expected.

As used herein, "differentiation" refers to the developmental process of Lineage commitment. "Lineage" refers to the pathway of cell development in which precursor or "ancestor" cells (in this invention, stem cells) undergo progressive physiological changes to become specific cell/organ types (in this invention, brain micro-organoids) with characteristic functions ).

As used herein, "enriched early brain micro-organ (population)" refers to purified or semi-purified micro-organ (population) of early brain micro-organ (population).

As used herein, the "IVD" refers to In vivo Developed Organoids, which construct brain organoids in vivo.

Preparation of early brain-like micro-organs

Based on the inventor's new discovery, the present invention provides a method for preparing brain-like micro-organs using pluripotent stem cells as starting cells, the method comprising: (1) culturing pluripotent stem cells to obtain embryoid bodies (EBs); (2) Cultivate the embryoid bodies of (1), induce them with induction medium (Induction medium), and obtain cultures containing early brain micro-organs (groups) of appropriate size, the diameter of the early brain micro-organs is 150-600 μm; preferably 200-500 μm; and (3) isolating early brain micro-organoids (populations) from the culture of (2).

In the present invention, in the preparation process of the "pluripotent stem cells", it is first necessary to make undifferentiated pluripotent stem cells form embryoid bodies, and the cultivation of embryoid bodies is a technique known to those skilled in the art. As a preferred mode of the present invention, the embryoid body is prepared by means of suspension culture. After obtaining the embryoid bodies, induce differentiation in the direction of brain micro-organs.

In the art, there are commercially available reagents (medium) for culturing/inducing pluripotent stem cells and obtaining embryoid bodies, and such reagents can be used in the present invention. As a preferred mode of the present invention, the medium for cultivating embryoid bodies is EB formation medium (purchased from Stem Cell Technologies); as a preferred mode of the present invention, the brain organoid induction medium is Induction medium (purchased from Stem Cell Technologies) ).

As a preferred mode of the present invention, the time for culturing pluripotent stem cells to obtain embryoid bodies is preferably 5±1 days, preferably 5±0.5 days. The time for culturing embryoid bodies to obtain early brain micro-organoids is preferably 24-72 hours, preferably 36-60 hours, more preferably 40-55 hours, and most preferably 48±2 hours. With such a culture time setting, a graft with a suitable size and an ideal post-transplant developmental state can be obtained.

In the present invention, "transplantation", "introduction" and "administration" can be used interchangeably, and refer to the method or approach that enables the early brain micro-organs (groups) prepared by the present invention to be positioned at the desired site, so that the Early brain micro-organoid(s) are placed in a subject, as an allogeneic or xenogeneic subject. The early brain micro-organoid (population) can be administered to the subject using a variety of suitable routes that result in delivery of the early brain micro-organ (population) to a desired site in the subject, where at least a portion of the early brain micro-organoid Stay alive. Preferably at least about 20%, preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 80%, most preferably at least about 85%, 90%, 95%, 98% or 99% % or more of the early brain micro-organoid(s) remain viable after administration to the subject. After administration to a subject, the early brain micro-organoids further develop and mature, and their vitality period can preferably be long-term, such as weeks, months or years. As a preferred mode of the present invention, the early organoid is transplanted into the brain by stereotaxic injection.

Using the culture method and culture medium of the present invention, it can be cultured in a two-dimensional culture system or a three-dimensional culture system. As a preferred mode of the present invention, culture is performed using a low-adsorption culture container. For example, all The culture container described above is a culture well plate, such as a 12, 24, 36, 48, 96, 192, 384 well plate.

As long as the isolation is performed at an appropriate time, the present invention has no particular limitation on the method of isolating early brain micro-organoids (populations), and conventional methods in the art can be used.

Traditional brain organoid transplantation uses mature brain organoids (usually large in size), which can easily cause damage to healthy brain tissue. Compared with the traditional method of transplanting mature brain organoids, the present invention innovatively injects early brain micro-organoids (with a suitable size) into the brain to be transplanted, which can continue to grow and differentiate in vivo, and produce mature Brain organoids (IVD brain organoids).

In some embodiments, the pluripotent stem cells described in the present invention may be pluripotent stem cells carrying a reporter gene or a tracer marker, which facilitates the observation of their state in vivo. Preferably, the reporter gene is introduced into the pluripotent stem cells by virus (such as lentivirus, adenovirus, adeno-associated virus) transfection. As a preferred mode of the present invention, the inventors have also optimized the virus transfection conditions, through the single-cell suspension transfection method; more preferably, the single-cell suspension transfection method is a single Cell suspension transfection) for virus transfection. Since hPSCs aggregate and grow in clumps, if virus transfection is carried out under ordinary adherent conditions, the transfection efficiency will be greatly reduced because only cells in the outer circle can be transfected. The present invention adopts a short-time single-cell suspension transfection method, which greatly improves It improves the transfection efficiency and avoids the cytotoxicity caused by co-incubation with the virus for a long time, and provides a new method for the tracer labeling and gene therapy of brain-like micro-organs. The reporter gene or tracer marker can use the reporter gene or tracer marker commonly used in the art, such as but not limited to GFP or EGFP and the like.

As a preferred mode of the present invention, in the technical solution of the present invention, the micro-organs are directly used for transplantation after being cultured and obtained, and the transplantation material is not coated with matrigel. However, the middle and late stages of traditional brain organoids need to be coated with matrigel. The matrigel layer covering the surface of the brain organoids will hinder the full contact and material exchange between the graft and the host tissue. If the treatment with matrigel removal reagents will inevitably reduce the Cell viability of grafts. The preparation process of the brain micro-organ in the present invention does not involve matrigel coating, which effectively avoids the above problems.

In the present invention, instead of transplanting large-sized mature brain organoids, the inventors innovatively injected smaller early brain micro-organoids into the mouse brain, allowing them to continue to grow and differentiate in vivo to produce mature Brain organoids (IVD brain organoids). Through immunostaining analysis, the inventors found that mature neuronal cells expressing MAP2 markers existed in IVD brain organoids. At the same time, compared with human brain organoids cultured in vitro, the inventors also found that more pericyte-like and mature cells were produced in IVD brain organoids. Choroid plexus (ChP) cells, which contribute to the maintenance of brain homeostasis. More importantly, these IVD brain organoids also showed reduced levels of cellular stress and apoptosis. Therefore, the results of the present inventors show that IVD brain organoid technology can effectively reduce the risk of surgical injury during brain organ transplantation, and it is expected to become a new type of cell therapy for neurological diseases.

Utilize the method of the present invention, can establish the system that mammalian pluripotent stem cell differentiates to early brain micro-organ (group), provide new research model for studying the molecular mechanism of directed differentiation of mammalian stem cell, be the clinical research and basis of micro-organ transplantation Research offers good avenues.

brain micro-organs

The present invention also includes the early brain micro-organ (group) obtained by the aforementioned preparation method, preferably, it is an isolated early brain micro-organ (group) that is easy to be used as a transplant. As a way of existence, in the micro-organ group, the number of early brain-like micro-organs accounts for more than 60% of the total number, preferably accounts for more than 70% of the total number; more preferably accounts for 80% of the total number, 85%, 90%, 95, 98% or more than 99%.

As a preferred mode of the present invention, after the early brain micro-organs (groups) are transplanted into the brain, they maintain a good active state, can continue to develop, and form mature brain organoids, which exhibit the following properties: include pericyte-like and mature choroid plexus cells (ChP epithelioid cells); neuronal differentiation occurs; astrocyte differentiation occurs; pericyte-like differentiation occurs; choroid plexus differentiation occurs. At the same time, the cell stress and/or apoptosis level of the early brain-like micro-organs (populations) is low, that is, the survival rate in vivo is very good.

Using FACS and surface marker technology, RT-PCR technology or other gene expression analysis technology, the marker characteristics presented by organs/tissues/cells can be analyzed, such as mature neuron marker MAP2, astrocyte marker GFAP, pericyte Labeled PDGFRβ, positive choroid plexus cells labeled TTR.

In the present invention, the starting cells/starting cells are pluripotent stem cells (such as pluripotent stem cells, embryonic stem cells or induced pluripotent stem cells) of mammals (human or non-human mammals), or are purchased from commercial sources, rather than Embryonic stem cells obtained by destroying an embryo. The starting cells/starting cells are preferably cells derived from humans, and may also be cells derived from non-human mammals (such as rabbits, mice, sheep, pigs, and monkeys).

The present invention also provides the use of the early brain micro-organoids for preparing a composition for brain transplantation, and the composition for brain transplantation is used for forming mature brain organoids, thereby: increasing the number of pericytes in the brain and mature choroid plexus cells; increased neuronal cells in the brain; increased astrocytes in the brain; increased pericytes in the brain; and/or increased choroid plexus cells in the brain.

The present invention also provides the use of the early brain micro-organoid for alleviating or treating brain damage; or for preparing a composition for brain transplantation, which is used for alleviating or treating brain damage.

The composition of the present invention includes a pharmaceutical composition, which contains an effective amount (such as 1 to 200, preferably 1 to 150; preferably 2 to 100; preferably 2 to 80; preferably 2 to 60; preferably 2-40; preferably 3-30; preferably 3-20; preferably 3-15; preferably 3-10; preferably 3-8 ; preferably 3 to 5; such as but not limited to 4, 6, 8, 9, 10, 12, 16, 18, 25, 35, 45, 55, 65, 75, 85, 95, 110, 120, 130 , 140, 150, 160, 180, 190, etc.) of the early brain micro-organoids of the present invention as active ingredients.

The early brain micro-organoid of the present invention can be mixed with an appropriate pharmaceutical carrier to prepare a pharmaceutical composition. The pharmaceutical composition is useful for groups in need (for example, patients with severe neuron loss or neurodegenerative diseases).

In the present invention, a "pharmaceutically acceptable" ingredient is a substance suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation and allergy), ie a substance with a reasonable benefit/risk ratio.

In the present invention, "pharmaceutically acceptable carrier (pharmaceutical carrier)" is a pharmaceutically acceptable solvent, suspending agent or culture solution for delivering early brain micro-organs to animals or humans.

The technical scheme of the present invention can effectively reduce the risk of surgical injury during brain organ transplantation, and the graft can produce more pericyte-like and mature choroid plexus (ChP) cells in IVD brain organoids, which is beneficial to brain homeostasis At the same time, these IVD brain organoids also showed reduced levels of cellular stress and apoptosis.

In terms of the volume of the graft, the early brain micro-organ obtained by the method of the present invention can effectively reduce the risk of surgical injury during brain organ transplantation, and is expected to become a new type of cell therapy for nervous system diseases.

From the perspective of the in vivo development of the graft, the early brain micro-organoids obtained by the method of the present invention exhibit more excellent in vivo performance than the organoids cultured for a long time in vitro, for example, they exhibit significantly more Pericyte-like and mature choroid plexus cells, pericytes, and choroid plexus cells when cultured in vitro at the same period; their caspase 3 levels (low apoptosis rate) were significantly lower than those of micro-organs cultured in vitro at the same period.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods that do not indicate specific conditions in the following examples, generally follow the conditions described in J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, or according to the manufacturer's suggestion conditions of.

Example 1. In vitro construction of early brain-like micro-organs

After digesting human pluripotent stem cells (hPSCs) into single cells with mild cell dissociation reagents, they were transfected with FUGW-GFP lentivirus for 1 hour (while adding 4 mg/ml polybrene) to obtain GFP labeling hPSC.

After transfection, the cells were resuspended with EB formation medium (Stem Cell Technologies), and added to a low-adsorption 96-well U-bottom plate at a density of 9000 cells per well.

After 5 days, the formed EBs were transferred to a low-adsorption 24-well plate, about 1-2 per well, and induction medium (Induction medium; Stem Cell Technologies) was added to continue culturing for 2 days.

The above-mentioned early brain micro-organoids (200-500 μm in diameter) cultured to the 7th day were washed with buffer solution (HBSS), sucked into a syringe and used for subsequent injection directly. After that, it was used in Example 2 for transplantation.

The early organoids at this stage have the following characteristics: morphologically, they have preliminarily formed organoid structures, and the cell composition is mainly composed of neural stem cells and precursors, which are different from mature brain organoids, which are mainly mature neurons.

Embodiment 2. In vivo construction of IVD brain organoids and staining detection method

In this example, the early brain micro-organoids obtained from the differentiation of GFP-labeled hPSCs obtained in Example 1 above were transplanted in vivo.

1. Stereotaxic injection

Mice were anesthetized with 125 mg/kg tribromoethanol (MedChemExpress) and placed in a stereotaxic apparatus. Drill holes in the skull to expose the dura mater, and three (because the main cells of the early brain micro-organs obtained above are composed of neural stem cells and precursor cells, which will continue to grow and differentiate after injection into the brain) early brain-like Microorganoids or isolated cells from the same number of digested microorganoids from earlier brain-like micro-organoids were injected with a 5 µl Hamilton syringe with a 22-gauge needle at the following coordinates: brain organoids, AP: +0.5 mm, ML: +2 mm; isolated cells, AP: +0.5 mm, ML: -2 mm. After injection, leave the needle in place for 2 minutes before removing it to avoid backflow.

2. Immunofluorescence staining

After PFA fixation, the mouse brain was soaked in 30% sucrose for 2-3 days until the sample sank. Mouse brains were then placed in OCT and cryosectioned at 30 μm thickness. Antigen retrieval was performed at 95° C. for 20 minutes with citrate antigen retrieval solution (Beyotime). Slides were incubated overnight at 4°C with primary antibodies diluted in PBS containing 0.3% Triton X-100 and 3% donkey serum. After three washes in PBS, slides were incubated with secondary antibodies for 1 hr at room temperature, three washes in PBS and co-stained with DAPI. The main antibodies used are as follows: MAP2 (rabbit anti, Millipore AB5622, 1:500), GFAP (rabbit anti, DAKO Z033401, 1:1000), STEM121 (mouse anti, Takara Y40410, 1:500), PDGFR (rat anti Thermofisher 14-1402-81, 1:500), Zbtb20 (rabbit anti-Proteintech 23987-1-AP, 1:200), TTR (mouse anti-R&D MAB7505, 1:500) The secondary antibodies used are as follows: donkey anti-rabbit cy3 (Jackson ImmunoResearch 711-165-152, 1:1000), donkey anti-mouse Alexa 647 (Jackson ImmunoResearch A31571, 1:1000), donkey anti-mouse cy3 (Molecular Probes 715-165-150, 1:1000), donkey anti- Mouse cy3 (Jackson ImmunoResearch 712-165-150, 1:1000). All images were scanned using an Olympus FV10i confocal microscope.

Embodiment 3, in vivo differentiation detection

The aforementioned GFP-labeled hPSC-differentiated early brain micro-organoids were cultured in vitro for 7 days, and these early organoids were transplanted into the striatum of immunodeficient mice by stereotaxic injection. As a control, the inventors also injected the same number of cells isolated from early brain micro-organoids into the striatum on the other side of the same mouse. The flow chart of the in vivo construction of IVD brain organoids is shown in Figure 1A.

After 8 weeks of transplantation, the inventors found that GFP-labeled IVD brain organoids can survive well in the host brain by analyzing the brain slices, and there are also pericyte-like and mature choroid plexus cells (ChP epithelial-like cells) ) surrounded by structures. The immunohistochemical staining diagram of the in vivo structure of IVD brain organoids is shown in Figure 1B.

The DAPI and STEM121 (human cell marker) staining of IVD brain organoids and equivalent isolated cells is shown in Figure 1C, while there was no staining in the contralateral brain striatum transplanted with isolated cells from early brain micro-organoids. This structure has been observed. This result indicates that this organoid-like structure is unique to IVD brain organoids.

The staining conditions of MAP2 and GFAP in IVD brain organoids are shown in Figures 1D and 1E. The inventors also detected mature neuron marker MAP2 and astrocyte marker GFAP, suggesting that neurons and astrocytes in IVD brain organoids cell differentiation.

Example 4, Cellular composition and state analysis of IVD brain organoids

The present inventors analyzed the cellularity and state of IVD brain organoids.

Through further immunostaining analysis, the inventors found that there were PDGFRβ-positive pericytes (Fig. 2A) and TTR-positive choroid plexus cells (Fig. 2B) in IVD brain organoids, both of which are important supporting cells. The maintenance of brain homeostasis plays an important regulatory role, and these two types of cells do not exist or are in small numbers in hCOs cultured in vitro.

Staining of cleaved caspase3 in IVD organoids and hCOs is shown in Figure 2C. The results showed lower levels of caspase 3 staining in IVD brain organoids compared with hCOs cultured in vitro, suggesting better survival.

Example 5. Comparison of in vitro construction of early brain micro-organs in different ways

Transfection was performed as described in Example 1 to obtain GFP-labeled hPSCs. After transfection, the cells were resuspended with EB medium (Stem Cell Technologies), and added to a low-adsorption 96-well U-bottom plate at a density of 9000 cells per well.

After 5 days, the formed EBs were transferred to a low-adsorption 24-well plate, and induction medium (Stem Cell Technologies) was added to continue culturing for 1 day, 2 days, 5 days, and 45 days. Observe the formed brain organoids; at the same time, transplant the co-cultured brain organoids into the body and observe their subsequent in vivo state. The results are shown in Table 1.

Table 1

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims. Also, all documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference.

Claims (16)

A method for preparing brain-like micro-organs, characterized in that the method comprises: (1) culturing pluripotent stem cells to obtain embryoid bodies; (2) culture the embryoid bodies of (1), induce them with neuroectodermal differentiation conditions, and obtain early brain micro-organ cultures containing suitable sizes, and the diameter of the early brain micro-organs is 150-600 μm; and (3) Isolation of early brain-like micro-organs from the culture of (2). The method according to claim 1, characterized in that, in step (2), the diameter of the early brain micro-organ is 200-500 μm. The method according to claim 1, characterized in that, in step (1), the culture time is 5±1 days; preferably 5±0.5 days. The method according to claim 1, characterized in that, in step (2), the culture time is 24 to 72 hours; preferably 36 to 60 hours; more preferably 40 to 55 hours; most preferably 48 hours ±2 hours. The method according to claim 1, characterized in that, in step (1), the culture medium is an embryoid body medium; preferably it is EB formation medium EB formation culture fluid. The method according to claim 1, characterized in that, in step (2), the culture medium is a brain organoid induction medium; preferably it is an induction medium induction differentiation medium. The method according to claim 1, characterized in that the culture is carried out in a culture container with low adsorption. The method according to claim 1, wherein, in (1), the pluripotent stem cells are pluripotent stem cells carrying a reporter gene or a tracer marker; preferably, the reporter gene is transfected with a virus Introduced into the pluripotent stem cells; more preferably, virus transfection is performed by a single cell suspension transfection method. The method according to claim 1, characterized in that, in (2), the step of matrigel coating is not included. The application of the method according to any one of claims 1 to 9 for preparing early brain micro-organoids. An early brain micro-organ, characterized in that it is prepared by the method described in any one of claims 1-9. The early brain-like micro-organ according to claim 11, wherein the early brain-like micro-organ forms a mature brain-like organ after being transplanted into the brain, preferably into the striatum, hippocampus, and cortex. Organs which exhibit the following properties: Contains structures surrounded by pericyte-like and mature choroid plexus cells; Neuronal differentiation occurs; Astrocyte differentiation occurs; pericyte-like differentiation occurs; Choroid plexus differentiation occurs; and/or Low levels of cellular stress and/or apoptosis. The early brain micro-organoid according to claim 12, wherein, in the differentiation of neurons, the brain organoid comprises neuronal cells expressing mature neuron marker MAP2; In astrocyte differentiation, the organoid comprises neuronal cells expressing the astrocyte marker GFAP; During pericyte differentiation, the organoid comprises PDGFRβ-positive pericytes; In the course of choroid plexus cell differentiation, the organoid comprises TTR-positive choroid plexus cells; and/or Said low level of cellular stress and/or apoptosis includes markedly low level of caspase 3 staining. The application of the early brain micro-organ according to any one of claims 10 to 13 for preparing brain transplantation With a composition, the composition for brain transplantation is used to form a mature brain organoid, thereby: Increased structures surrounded by pericyte-like and mature choroid plexus cells in the brain; Increase neuron cells in the brain; Increased astrocytes in the brain; Increased pericytes in the brain; and/or Increased choroid plexus cells in the brain. The application of the early brain micro-organoid according to any one of claims 10 to 13 is used for preparing a composition for brain transplantation, and the composition for brain transplantation is used for alleviating or treating brain damage. A composition for brain transplantation, characterized in that it comprises: The early brain micro-organ according to any one of claims 10-13; and pharmaceutically acceptable carrier.