阅读说明：本技术 用于产生生物工程化的神经元类器官(beno)的方法及其用途 (Methods for producing bioengineered neuronal organoids (BENO) and uses thereof ) 是由 W-H·齐默尔曼 M·P·扎菲里奥 于 2018-06-08 设计创作，主要内容包括：本发明涉及神经组织(特别是大脑)的体外3D建模领域。需要开发反映神经组织的生理方面的神经组织的细胞培养模型。本发明提供了产生形成功能性神经元网络的生物工程化的神经元类器官(BENO)的方法。本发明还涉及例如在药物筛选和个性化药物领域中所产生的BENO的用途和应用。(The present invention relates to the field of in vitro 3D modeling of neural tissue, in particular the brain. There is a need to develop cell culture models of neural tissue that reflect physiological aspects of neural tissue. The present invention provides methods for generating bioengineered neuronal organoids (BENO) that form functional neuronal networks. The invention also relates to the use and application of the produced BENO, for example in the field of drug screening and personalized medicine.)

1, A method of producing a bioengineered neuronal organoid (BENO) from Pluripotent Stem Cells (PSCs), the method comprising:

(A) providing a source of PSCs;

(B) culturing the PSCs of step (a) embedded in a matrix submerged in a cell culture medium;

(C) culturing PSCs in the matrix of step (B) in a cell culture medium comprising a Rho-associated kinase inhibitor (ROCKi) and FGF-2;

(D) culturing the PSC and matrix-forming BENO derived from step (C) in a cell culture medium comprising retinoic acid and or more SMAD signaling inhibitors to induce neurogenesis;

(E) culturing the BENO formed in step (D) in a cell culture medium comprising TGF- β and FGF-2 to enhance stromal cell genesis and neurogenesis;

(F) culturing the BENO formed in step (E) in a cell culture medium comprising TGF- β and or more notch signaling inhibitors to enhance the development and neural differentiation of stromal cells.

2. The method of claim 1, wherein the matrix does not comprise Matrigel.

3. The method of claim 1, wherein the matrix does not comprise Matrigel or other ingredients of natural origin with undefined components.

4. The method according to claims 1-3, wherein the matrix comprises collagen, preferably type I collagen.

5. The method of claims 1-4, wherein BENO is generated within a 3D environment, preferably wherein the 3D environment is defined by a substrate.

6. The method of claims 1-5, wherein the stromal cells comprise glial cells.

7. The method of claims 1-6, wherein the mediator of step (D) comprises at least two inhibitors of SMAD signaling, preferably wherein the inhibitors of SMAD signaling comprise noggin and SB 431542.

8. The method of claims 1-7, wherein the notch signaling inhibitor of step F is DAPT.

9. The method of claims 1-8, wherein the PSCs are human PSCs.

10. The method of any one of claims 1-9,

wherein step (A) and step (B) are performed on day-1,

wherein step (C) is performed on days-1 to 0,

wherein step (D) is performed from day 0 to day 10,

wherein step (E) is performed from day 10 to day 15,

wherein step (F) is performed from day 15 to at least day 28.

A neuron organoid of 11, , characterized in that the neuronal cells of said neuron organoid are organized in a functional neuronal network.

12, bioengineered neuronal organoids (BENO) produced by the method of claims 1-11.

13. Use of BENO according to claim 11 or 12 as a model for disease and/or drug screening.

14. The use of claim 13, wherein the BENO is co-cultured with other tissue engineering platforms.

15, a kit for performing the method of claims 1-9.

Background

In vitro 3D modeling of neural tissue, particularly brain tissue derived from mammals (e.g., human brain), represents powerful tissue bioengineering tools that can be used to study complex neuronal cell systems in general, 3D cell culture systems provide faster cell differentiation, higher cell complexity and longevity than corresponding 2D culture systems promising methods for in vitro 3D modeling of neural tissue include the use of Pluripotent Stem Cells (PSCs), which have been used in the modeling of various human tissues and organs, these advantages of 3D modeling methods in combination with recently used techniques (e.g., reprogramming patient fibroblasts to induced PSCs) collectively provide a new field of view for elucidating potential molecular mechanisms responsible for a variety of human neuronal diseases.

such methods include induction of neuroectoderm by dual SMAD (Sma and Mad Related Family) signaling pathway inhibition (i.e., inhibition by BMP and TGF β) in culture for about 8-12 days (Chambers, Fasano et al, nat. biotechnol, 2009). at this point in time, most stem cells are transformed into Neural Progenitor Cells (NPC). after 12 days, several approaches (Lancaster and Knoblich, Science,2014) allow cells to spontaneously differentiate into various neurons and glial cells, while others (Qian, Nguyen et al, Cell, 2016; Birey, Andersen et al, Nature,2017) apply various pattern factors to model tissues or apply neurotrophic factors (BDNF, GDNF) to enhance neuronal survival.

Despite the advances in the generation of human neuronal organoids, there are still a number of drawbacks that limit the utility of existing neuronal organoids. For example, known methods lack precise definition of neuronal organoids; thus, such organoids often exhibit high phenotypic variability. This is due in part to the conventional use of Matrigel as a neurogenesis-supporting substrate.

Another disadvantages associated with known methods is that the resulting organoids lack neuronal network function, thus significantly limiting any study of neuronal function and plasticity due to the remote phenotypic similarity of the known resulting neuronal organoid structures compared to normal brain tissue, disease modeling and drug development is also limited.

Additionally, there is a need to generate neuron organoids capable of forming a functional neuronal network to meaningfully mimic natural neural structures.

The present invention provides a method that allows robust and reproducible neural differentiation in well-defined 3D cell culture systems, which further step provides a good basis for studying the formation and plasticity characteristics of functional neuronal networks.

Additionally, new biologies (e.g., non-coding RNA therapeutics) and genome editing (e.g., using CRISPR-based platforms) can be effectively tested in human models.

Disclosure of Invention

The present invention relates to methods for producing a bioengineered neuronal organoid (BENO) from Pluripotent Stem Cells (PSCs), the method comprising:

(A) providing a source of PSCs;

(B) culturing the PSCs of step (a) embedded in a matrix submerged in a cell culture medium;

(C) culturing the PSCs in the matrix of step (B) in a cell culture medium comprising a Rho-associated kinase inhibitor (ROCKi) and FGF-2;

(D) culturing the PSC and matrix-forming BENO derived from step (C) in a cell culture medium comprising retinoic acid and or more SMAD signaling inhibitors to induce neurogenesis;

(E) culturing the formed BENO of step (D) in a cell culture medium comprising TGF- β and FGF-2 to enhance stromal cell genesis and neurogenesis;

(F) culturing the formed BENO of step (E) in a cell culture medium comprising TGF- β and or more notch signaling inhibitors to enhance the development and neural differentiation of stromal cells.

In some embodiments the matrix does not comprise Matrigel in other embodiments the matrix does not comprise Matrigel or other components of natural origin having undefined components in preferred embodiments the matrix comprises collagen in most preferred embodiments the matrix comprises type I collagen in some embodiments the matrix is collagen in some embodiments the matrix is collagen I in some embodiments the matrix is collagen I.

In embodiments, the BENO is generated within a 3D environment, preferably wherein the 3D environment is defined by a substrate.

In some embodiments of , the stromal cells comprise glial cells.

In some embodiments of , the mediator of step (D) comprises at least two inhibitors of SMAD signaling, preferably wherein the inhibitors of SMAD signaling comprise noggin and SB431542 in some embodiments of , the inhibitors of SMAD signaling are noggin and SB 431542.

In embodiments, the notch signaling inhibitor of step F is DAPT.

In embodiments, the SMAD signaling inhibitor is noggin and SB431542, and the notch signaling inhibitor of step F is DAPT.

In some embodiments of , the matrix is collagen, which is used at a concentration of 0.05mg/ml to 50mg/ml, preferably 0.1mg/ml to 10mg/ml, more preferably 0.5mg/ml to 5mg/ml, and most preferably 1 mg/ml.

In embodiments retinoic acid is used at an effective concentration of 0.01 μ M to 100 μ M, preferably 0.1 μ M to 10 μ M, more preferably 0.5 μ M to 5 μ M, most preferably 1 μ M in embodiments noggin is used at an effective concentration of 0.1ng/ml to 1 μ g/ml, preferably 1ng/ml to 500ng/ml, more preferably 10ng/ml to 200ng/ml, most preferably 50ng/ml in embodiments SB431542 is used at an effective concentration of 0.1 μ M to 1mM, preferably 1 μ M to 100 μ M, more preferably 5 μ M to 50 μ M, most preferably 10 μ M in embodiments TGF- β is used at an effective concentration of 0.1ng/ml to 100ng/ml, preferably 0.3ng/ml to 30ng/ml, more preferably 1ng/ml to 10ng/ml, most preferably 5ng/ml to 5 μ M, most preferably 0.1 μ M to 5 μ M, most preferably 0.1ng/ml to 10 μ M in1 μ M to 100 μ M, preferably 0.3ng/ml to 30ng/ml, more preferably 1 μ M to 10ng/ml, most preferably 5 μ M to 5 μ M, most preferably 1ng/ml, preferably 0.1 μ M to 10 μ M to 5 μ M to 1mg/ml, preferably 0.5 μ M to 1 to 10 μ M to 1 to 5 μ M to 1 to 5 μ M to 1 to 5 μ M.

In some embodiments of , retinoic acid is used at a concentration of 0.5 μ M to 5 μ M, noggin is used at a concentration of 10ng/ml to 200ng/ml, SB431542 is used at a concentration of 5 μ M to 50 μ M, TGF- β is used at a concentration of 1ng/ml to 10ng/ml, FGF-2 is used at a concentration of 5ng/ml to 50ng/ml, and DAPT is used at a concentration of 0.5 μ M to 5 μ M.

In embodiments, retinoic acid is used at a concentration of 0.5 μ M to 5 μ M, noggin is used at a concentration of 10ng/ml to 200ng/ml, SB431542 is used at a concentration of 5 μ M to 50 μ M, TGF- β is used at a concentration of 1ng/ml to 10ng/ml, FGF-2 is used at a concentration of 5ng/ml to 50ng/ml, DAPT is used at a concentration of 0.5 μ M to 5 μ M, and matrix is collagen used at a concentration of 0.1mg/ml to 10 mg/ml.

In some , retinoic acid is used at a concentration of 1 μ M, noggin is used at a concentration of 50ng/ml, SB431542 is used at a concentration of 10 μ M, TGF- β is used at a concentration of 5ng/ml, FGF-2 is used at a concentration of 10ng/ml, and DAPT is used at a concentration of 2.5 μ M.

In some embodiments of , retinoic acid is used at a concentration of 1 μ M, noggin is used at a concentration of 50ng/ml, SB431542 is used at a concentration of 10 μ M, TGF- β is used at a concentration of 5ng/ml, FGF-2 is used at a concentration of 10ng/ml, DAPT is used at a concentration of 2.5 μ M, and matrix is collagen used at a concentration of 0.5mg/ml to 5 mg/ml.

In some embodiments the PSC is an animal cell in some embodiments the PSC is a mammalian cell in some embodiments the PSC is a rodent (e.g., mouse or rat) or human cell in some embodiments the PSC is a human PSC.

The various steps of the present invention are performed at different times in embodiments, step (A) and step (B) are performed on days-1, in embodiments, step (C) is performed on days-1 to day 0, in embodiments, step (D) is performed on days 0 to day 8, in embodiments, step (E) is performed on days 8 to day 15, in embodiments, step (F) is performed on days 15 to at least day 28, in embodiments, step (A) and step (B) are performed on days-1, step (C) is performed on days-1 to day 0, in embodiments, step (A) and step (B) are performed on days-1, step (D) is performed on days 0 to day 8, step (A) and step (B) are performed on days-1, step (C) is performed on days-1 to day 0, step (D) is performed on days 0 to day 8, step (C) is performed on days 350 to day 0, step (C) is performed on days 0 to day 10, step (D) is performed on days 0, step (C) is performed on days 1 to day 10, step (D) is performed on days, step (C) to day 10) is performed on days, step (D) is performed on days 1 to day 10) is performed on days, step (D) is performed on days 0) is performed on days, step (D) is performed on.

the invention provides a neuron organoid, such as a bioengineered neuron organoid (BENO), characterized in that the neuronal cells of the neuron organoid are organized in a functional neuronal network the invention provides a bioengineered neuron organoid (BENO) produced by the method of the invention.

In some aspects , the invention relates to the use of BENO produced by the methods of the invention as a disease model in some embodiments , the invention relates to the use of BENO produced by the methods of the invention as a disease model associated with neural tissue in some embodiments , the invention relates to the use of BENO produced by the methods of the invention as a disease model selected from stroke, encephalitis disorders, neurodegenerative diseases, neuroinflammatory diseases, traumatic injury, ion channel diseases, and psychosis in some embodiments , the invention relates to the use of BENO produced by the methods of the invention as a disease model selected from neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease), neuroinflammatory diseases (such as multiple sclerosis), traumatic injury (such as that caused by brain surgery), ion channel diseases (such as epilepsy), and psychosis (such as autism, schizophrenia).

At , the other tissue-engineered platforms are selected from EHM (engineered myocardium), BSM (bioengineered skeletal muscle), ESM (engineered skeletal muscle), ELT (engineered liver tissue), and ECT (engineered connective tissue).

In some aspects , the present invention relates to the use of BENO produced by the methods of the present invention in drug screening, such as drug discovery and drug refinement by phenotypic drug screening.

In other aspects, the invention relates to kits for performing the methods of the invention in embodiments the kit comprises a PSC, a matrix, a suitable medium, and a desired supplement (ROCKi, FGF-2, retinoic acid, inhibitors of one or more SMAD signaling of , inhibitors of TGF- and 0 or more notch signaling). in other embodiments the kit comprises a matrix, a suitable medium, and a desired supplement (ROCKi, FGF-2, retinoic acid, inhibitors of one or more SMAD 387 signaling of , inhibitors of one or more notch signaling of TGF-585 and ).

Drawings

FIG. 1 is a typical protocol for neural differentiation from stem cells.

Figure 2 enhanced neurogenesis under dual SMAD signaling pathway inhibition. Fig. 2A, IF analysis of tissue treated with noggin or noggin and SB431542 only. Neurons were visualized using antibodies against neurofilaments (FITC-green) and nuclei with DAPI (blue). The histogram shows 10 μm. The right panel shows quantification of the mean fluorescence ratio from the whole tissue. Figure 2B, qPCR analysis of the neuronal markers PAX6 and MAP2 showed that both markers increased 21-fold after SB431542 treatment (n-4 tissues/group).

FIG. 3FGF-2 enhances neuronal differentiation. FIG. 3A, increased pluripotent stem cell input resulted in enhanced neurofilament staining at day 28 of BENO culture. FIG. 3B, proliferative effects of FGF-2 on stem cells and NPC. Higher numbers of neurons within the tissue and higher network complexity were observed with neurofilament staining in FGF-2 treated tissues on days 8-15.

FIG. 4 neuronal transcript analysis after BDNF and GDNF treatment. From day 10 to day 28, the expression of both PAX6 and MAP2 was not enhanced after BDNF and GDNF were added to the culture. BENO was analyzed on day 28.

Figure 5 enhances neurogenesis by notch inhibition. DAPT treatment from day 15 to day 28 increased the abundance of PAX6 transcript, thus indicating the presence of a higher number of neurons (commit).

FIG. 6A, summarizes the treatment protocol performed in each protocol of example 1 FIG. 6B, transcriptome time course analysis of BENO production OCT4 was used as a stem cell marker, GFAP was used as a glial marker, PAX6 was used as an NPC and neuronal marker, and MAP2, GRIN1 and GABBR2 were used as mature neuronal markers data was normalized to GAPDH FIG. 6C, embedding of whole tissue specimens of BENO (whole mount) IF analysis was performed on day 60, neurofilament, MAP2, Synaptophysin (Synaptophysin) and GFAP were used to stain neurons, mature neurons, synapses and glia, respectively FIG. 6D, BENO activity calculated by measuring calcium activity recorded in 5 regions of tissue.

Figure 7 heatmap of RNAseq analysis over time of development showing RNAseq data for neurogenesis and maturation in BENO. Fig. 7A, depicts markers of different states of stem cell differentiation into neurons and glial cells. Fig. 7B, markers for different neuron identities. Fig. 7C, cortical layer markers. FIG. 7D, different mature proteins as receptors, ion channels and synapse-associated proteins. Completed in cooperation with professor Rashi Haider/a.fischer (DZNE).

FIG. 8 cortical layer development in BENO. TBR2⁺The ependymal progenitor cells migrate concentrically from the middle of the organoid to the periphery. There, CTIP2 labels deep neurons.

FIG. 9BENO contains inhibitory and excitatory neurons. FIG. 9A, GABA is strongly expressed in the perikaryon (center) and synaptic nodes of GABAergic neuronal axons (periphery). Fig. 9B, the GABBR2 receptor was found to be expressed in the neuronal perikarya (center). FIG. 9C, Tyrosine Hydroxylase (TH) -labeled dopaminergic neurons were found in and around organoids similar to the GABA site. FIG. 9D, synaptophysin staining, indicates the presence of a very tight synaptic network around the organoid. Neurofilaments, MAP2 and DAPI were used to stain axons, mature neurons and nuclei, respectively. Bar chart: and 10 μm unless otherwise specified.

Neuronal network function in the BENO of FIG. 10 indicates integrated and hierarchical synaptic function. The left panel shows Fura-4 stained neurons. Matlab analysis of the different regions of interest (ROI) showed 12 different traces shown in the right panel. Before GABAR suppression, ROI (2,3), ROI (4,5), ROI (6, 7) and ROI (11,12) are synchronized. GABAR inhibition caused the cells to be out of sync, and after 10 minutes of washing, the cells were re-synchronized. Completed in cooperation with professor Guobin Bao/d.schild (UMG).

Figure 11 optimized duration of incubation with NCM and NPEM. Figure 11A, qPCR analysis of relative PAX6 transcript expression was performed 15 days after BENO production using the procedures indicated in the protocol. Fig. 11B, the maximum duration of the BENO treatment using NCM is defined (step D). qPCR analysis of relative PAX6 transcript expression was performed 15 days after the production of BENO using the procedures indicated in the protocol. FIG. 11C, immunofluorescence analysis of PAX6/ki67 positive cells to label proliferative neuronal progenitor cells.

Detailed Description

Definition of

As used herein, the term "organoid" refers to a tissue culture that forms a three-dimensional assembly that at least partially mimics the structure and/or function of an organ (e.g., a human organ). The organoids can be generated from pluripotent stem cells in, for example, a three-dimensional (3D) environment. such 3D environments for organoids are spherical 3D environments.

As used herein, the term "bioengineered neuronal organoids" (BENO) is an organoid derived from neural tissue produced according to the methods of the invention. BENO can be considered a miniaturized and simplified model of neural organs (including the brain) or neural tissue present within or controlling organs, such as neural tissue present in the heart (e.g., the sympathetic nervous system) and skeletal muscle (e.g., the nicotinic nerve endings at the skeletal neuromuscular junction).

As used herein, "BENO formed" is a composition of cells and matrix in the process of developing into BENO. The formed BENO is characterized in that its cells and matrix material have undergone step C of the method of the invention, but not yet undergone step F of the method of the invention or not yet completed.

In the case of cell culture, the 3D environment corresponds to structures in which the cells are arranged in three dimensions with respect to each other. examples of 3D environments are spherical arrangements.unlike the 3D environment is a 2D environment in which the cells are arranged in a monolayer, for example, there is no difference in the dimension of the spatial relationship between the cells.

As used herein, the term "3D cell culture system" refers to a cell culture at least initially in a 3D environment defined by a 3D matrix.

PSCs can be derived from a number of sources, including but not limited to Induced Pluripotent Stem Cells (iPSCs), parthenogenetic stem cells, stem cells resulting from nuclear transfer, and Embryonic Stem Cells (ESCs), and combinations thereof.

As used herein, the term "reprogramming" refers to methods in which a more specialized -ized cell or some other form of cell at an advanced stage of development can be converted into a pluripotent cell.

As used herein, the term "differentiated cell" refers to a cell that has developed from a precursor phenotype that is not exclusively -committed to a phenotype that is exclusively -committed.

As used herein, "induced pluripotent stem cells" (ipscs) refer to pluripotent stem cell types artificially derived from non-pluripotent cells, typically adult cells for example, induced pluripotent stem cells are considered similar, if not identical, to natural pluripotent stem cells (including embryonic stem cells) in terms of expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling times, embryoid body formation, teratoma formation, feasible chimera formation and potential and differentiability.

As used herein, the term "neural progenitor cell" (NPC) refers to a cell derived from a PSC that has the ability to proliferate to regenerate itself in exact copies (self-renewal) and to produce cell progeny of uniquely differentiated cells. The progeny of an NPC may be a neuronal cell (e.g., a neuronal precursor or mature neuron) or a glial cell (e.g., a glial cell precursor, mature astrocyte or mature oligodendrocyte). NPC, when cultured in vitro by themselves, typically do not produce progeny of other embryonic germ layers unless, for example, they are somehow dedifferentiated or reprogrammed. Unlike stem cells such as PSC, NPC has a limited ability to proliferate, and thus does not exhibit a self-sustaining ability.

The term "Matrigel" as used herein is a composition derived from the sarcoma of Engelbreth-Holm-Swarm mice (Kleinman et al, Biochemistry, 1982.) Matrigel is mixtures that are not well defined chemically, but generally contains laminin, collagen IV, heparan sulfate proteoglycans, entactin, and growth factors.

The term "chemically defined" or "definitively" as used herein refers to a composition whose chemical composition is known to a sufficiently precise degree. For example, up to 10%, preferably up to 5% of the total content of the composition is chemically uncharacterized or varies among different samples of the composition.

As used herein, "a component of natural origin having an undefined composition" relates to a composition (cell or tissue from animal, plant, fungal and protozoan cells or viruses) isolated from a natural source, which is not chemically defined precisely. For example, at least 10% of the total content of the composition must be uncharacterized or varied in different samples of the composition. Examples of naturally derived components having "indeterminate composition" include serum and Matrigel.

As used herein, the term "fibroblast growth factor-2" (FGF-2) is a member of the fibroblast growth factor family. FGF-2 is encoded by the FGF2 gene. FGF-2 is also known as "basic fibroblast growth factor (bFGF)". The terms FGF-2 and bFGF are used interchangeably herein.

As used herein, a "neuron network" represents groups or more interconnected neurons, where the connections between neurons allow information to be transmitted from neurons to additional neurons.

As used herein, "neuronal network organization" refers to the organization of groups of neurons as a network of neurons.

As used herein, a "functional neuronal network" refers to a neuronal network that displays electrochemical information transmission from neurons to another neurons.

As used herein, "neuronal network function" is the transmission of electrochemical information from neurons to another neurons.

As used herein, the term "induce" refers to the initiation and/or enhancement of a particular physiological effect, such as cell proliferation or cell differentiation.

As used herein, "neurogenesis" refers to differentiation and/or proliferation of cells into fully differentiated neural cells that cannot be differentiated by step . thus, neurogenesis includes differentiation of PSCs into NPCs, proliferation of NPCs, differentiation of NPCs into more differentiated neural cells, e.g., differentiation into neuronal cells (such as neuronal precursors or mature neurons) or glial cells (such as glial cell precursors, mature astrocytes or mature oligodendrocytes), or proliferation of more differentiated neural cells.

Thus, neural differentiation includes the differentiation of NPCs into more highly differentiated neural cells, such as neuronal cells (e.g., neuronal precursors or mature neurons) or glial cells (e.g., glial cell precursors, mature astrocytes or mature oligodendrocytes), or the differentiation of more highly differentiated neural cells into even more differentiated neural cells, e.g., from neuronal precursors into mature neurons.

As used herein, "encapsulation of cells in a matrix" refers to the interaction of cells with the matrix and/or the attachment of cells to the matrix. This process is regulated by cell-matrix interactions, for example by the regulation of cell receptors including integrins.

As used herein, the term "matrix" refers to a material that can create a 3D environment suitable for embedding cells. Preferably, the matrix of the invention forms a hydrogel structure. Exemplary suitable matrices include collagen or synthetic collagen mimetics.

As used herein, "hydrogel" refers to a network of hydrophilic polymers that includes water, but which is not water-soluble. The hydrogel molecules are chemically and/or physically linked, for example by covalent or ionic bonds or entanglements, to form a 3D environment. The hydrogel network may also be a natural or synthetic polymer network.

As used herein, the term "stromal cell" refers to a neural cell that is not a neuronal cell. In particular, exemplary stromal cells include glial cells, such as glial cell precursors, mature astrocytes or mature oligodendrocytes.

As used herein, the term "signaling" refers to the transmission of information (signals) within a cell or between two or more cells. Signaling can occur through chemical reaction means (e.g., phosphorylation, protein cleavage), through the release of signaling molecules (including ions, neurotransmitters), or through changes in direct electrochemical potential.

As used herein, "tissue engineered platform" refers to the in vitro assembly of cells designed to mimic the structural and/or functional characteristics of a tissue, preferably a human tissue.

As used herein, the term "phenotypic drug screening" refers to screening for the suitability of a new or existing drug based on its effect on the phenotype of a model system.

As used herein, the terms "bioengineered" and "bioengineered" refer to methods of manipulating biological systems and biological materials. Examples of bioengineering are molecular cloning, transfection, transduction, and the use of chemicals or other substances to affect cells.

As used herein, the term "disease modeling" refers to methods of generating a disease model that at least partially mimics some or all of the characteristics of a disease.

All terms not specifically defined herein are to be understood according to their customary meaning in the biological and medical fields, in particular in the field of stem cell and organoid research.

Principle of the invention

The present invention provides a method of producing bioengineered neuronal organoids (BENO) under chemically defined conditions which is reproducible and produces -derived products retinoic acid and FGF-2 for enhancing neurogenesis in the presence of TGF- β for supporting neurogenesis, representing a unique combination of biological activities of neurogenesis in the stromal environment leading to the formation of a functional neuronal network, exemplary suitable matrices of the invention are collagen hydrogels.

The method of the present invention is developed through several iterations, e.g., as disclosed in example 1, which results in a structure that exhibits significant neuronal network organization and function, which is supported by co-developing stromal cells such as glial cells the network organization and function, e.g., the formation of functional synapses between different neuronal cells of the neuronal organoids disclosed herein (thus, the functional ranking as described in example 5), provides numerous advantages over conventional organoid structures.

The presently disclosed method of the present invention includes a number of implementation steps. The steps are as follows:

(A) providing a source of PSCs;

(B) culturing the PSCs of step (a) embedded in a matrix submerged in a cell culture medium;

(C) culturing the PSCs in the matrix of step (B) in a cell culture medium comprising a Rho-associated kinase inhibitor (ROCKi) and FGF-2;

(D) culturing the PSC and matrix-forming BENO derived from step (C) in a cell culture medium comprising retinoic acid and or more SMAD signaling inhibitors to induce neurogenesis;

(E) culturing the formed BENO of step (D) in a cell culture medium comprising TGF- β and FGF-2 to enhance stromal cell genesis and neurogenesis;

(F) culturing the formed BENO of step (E) in a cell culture medium comprising TGF- β and or more notch signaling inhibitors to enhance the development and neural differentiation of stromal cells.

Providing Pluripotent Stem Cells (PSC) (step A)

The present invention relates to the production of bioengineered neuronal organoids (BENO) from Pluripotent Stem Cells (PSCs). Pluripotent stem cells may be obtained from a variety of sources, including, but not limited to, induced pluripotent stem cells (ipscs) (which may be generated by reprogramming cell types including fibroblasts, keratinocytes, bone marrow-derived cells, or blood-derived cells (e.g., umbilical cord blood-derived cells)), parthenogenetic stem cells, stem cells generated by nuclear transfer, and embryonic stem cells and/or mixtures thereof. The PSCs of the present invention are not produced in methods involving methods for modifying the genetic characteristics of human germline or human embryo applications involving industrial or commercial purposes. The methods of the invention can also be performed using a PSC cell line, such as the iPSC-G1 cell line described in Tibury et al, Circulation, 2017.

PSCs are characterized by the property of self-replication in the mass-expression of sternness factors in the undifferentiated state (e.g., Oct-3/4, SSEA-4, and TRAl-60) and the propensity to differentiate into tri-germ layer cells (endoderm, ectoderm, and mesoderm). The PSC may also be an induced PSC (ipsc).

Prior to use in the presently claimed methods, the PSCs are cultured under suitable conditions known in the art. If desired, PSCs can be cultured according to standard maintenance procedures, e.g., grown on a maintenance support such as Matrigel. The PSCs are grown in any suitable cell culture medium known in the art. An exemplary cell culture medium is TeSR-E8 base medium (Stemcell), which optionally comprises a Rho-associated protein kinase inhibitor (ROCki), for example at a concentration of 5. mu.M or 10. mu.M. Exemplary culture Methods for PSCs are reported in storer and Schwartz, Methods Mol biol.2011.

When the PSC is used in the present invention, the PSC is separated from its maintenance support such as Matrigel, for example, by EDTA treatment. EDTA may be used in a concentration of 0.1-10mM, preferably 0.5-2 mM. The EDTA treatment is carried out for 1 to 10 minutes, preferably 4 to 6 minutes. The preferred EDTA treatment conditions are 0.5mM EDTA for 5-10 minutes at room temperature and 2mM EDTA for 5-8 minutes at room temperature.

Culturing PSCs embedded in a matrix submerged in cell culture medium ("step B")

The PSCs provided above are then cultured with a matrix that allows the PSCs to be embedded in a 3D environment defined by the matrix. The embedding and self-organization of PSCs in such a 3D environment provides the basis for organoids of the present invention. Preferably, the cells should be embedded in the matrix in a uniform manner.

The matrix of the present invention constitutes a 3D environment that promotes self-organization and differentiation of PSCs and cells derived from PSCs. The structure of the matrix is stabilized by interactions of individual matrix molecules (e.g., protein-protein interactions). Most preferably, the matrix of the invention forms a hydrogel structure.

Exemplary suitable matrices for the present invention are collagen, collagen mimetics, alginate, fibrin, Matrigel, and chitosan. Preferred matrices are collagen and collagen mimetics. The most preferred matrix is based on type I collagen. The type I collagen-based matrix may contain trace amounts of other collagens, such as type III collagen. Preferably, the type I collagen-based matrix comprises greater than 80%, greater than 85%, greater than 90%, greater than 95% or 100% type I collagen.

In preferred embodiments, the matrix of the invention does not comprise Matrigel in a more preferred embodiment, the matrix of the invention does not comprise Matrigel or other components of natural origin having an undefined or undefined composition of components this point is particularly important because the use of undefined or undefined chemical mixtures (such as Matrigel) inevitably leads to large phenotypic differences in the resulting organoids, for example this observation is demonstrated in Tiburcy et al Circulation,2017, Matrigel is a well known undefined mixture comprising laminin, collagen IV, heparan sulfate proteoglycans, nestin and growth factor components derived from Engelbreth-Holm-Swarm mouse sarcoma, where the percentage of components between batches shows high variability (kleinbman et al Biochemistry, 1982).

The PSC and matrix elements are mixed in a cell culture medium suitable for PSC culture, examples of such cell culture media are TeSR-E8 base medium (Stemcell) and StemFlex medium (Gibco). the cell culture medium can be supplemented with other components, such as FGF-2 and ROCKi, which can enhance cell survival and proliferation in the matrix. exemplary media are TeSR-E8 base medium supplemented with 20ng/ml FGF-2 and 10. mu.M ROCKi.

In some embodiments , the matrix is at a concentration of 0.05mg/ml and 50 mg/ml. in other preferred embodiments, the matrix is at a concentration of 0.1mg/ml to 10 mg/ml. in more preferred embodiments, the matrix is at a concentration of 0.5mg/ml to 5 mg/ml. in most preferred embodiments, the matrix is at a concentration of 1 mg/ml.

The density of the PSC, after mixing with the medium and the matrix, can be from 0.1 to 10X 10 per ml⁶Range of individual cells. A preferred range is 0.5 to 6X 10 per ml⁶And (4) cells. More preferred range is 1-4X 10 per ml⁶ exemplary suitable values are 3X 10 per ml⁶And (4) cells.

This culturing step is performed before the culturing step disclosed in the following section. This culturing step (in which the PSC is embedded in a matrix; step B) is carried out before a culturing step of culturing cells in a cell culture medium comprising a Rho-associated kinase inhibitor (ROCKi) and FGF-2 ("step C"). The step B to step C transition is generally characterized by the addition of the ingredients of step C to the existing cell culture medium.

In some embodiments of , the culturing step B is conducted for a period of time of 1 minute to 1 day, in preferred embodiments step B is conducted for a period of time of 5 minutes to 5 hours, in more preferred embodiments step B is conducted for a period of time of 10 minutes to 1 hour, in even more preferred embodiments step B is conducted for a period of time of 15 minutes to 30 minutes, in most preferred embodiments step B is conducted for a period of time of 20 minutes.

In the protocol relating to the production of organoids of the invention, the time point of step B is referred to as "day-1".

Step B is carried out in a suitable cell culture vessel. An exemplary suitable cell culture vessel is a 96-well plate with a U-shaped bottom and low adhesion properties.

Step B is performed under conditions suitable for PSC survival. Exemplary suitable conditions are 37 ℃, 5% CO₂For example in a cell incubator.

Culturing said of step B in a cell culture medium comprising a Rho-associated kinase inhibitor (ROCKI) and FGF-2 PSC in a matrix ("Step C')

Culturing the PSCs in the matrix of step B in a culture medium comprising a Rho-associated kinase inhibitor (ROCKi) and fibroblast growth factor-2 (FGF-2).

Suitable rock ki variants include Y27632(Stemgent), Fasudil, ripassdil, RKI-1447, GSK429286A, Y-30141, and other components as reviewed in Feng et al, J Med chem., 2016. rock ki is used in effective concentrations a preferred rock ki is y27632. in some embodiments , Y27632 is at a concentration of 0.1 μ M to 1 mM.. in preferred embodiments, Y27632 is at a concentration of 1 μ M to 100 μ M. in more preferred embodiments, Y27632 is at a concentration of 5 μ M to 50 μ M. in most preferred embodiments, Y27632 is at a concentration of 10 μ M.

In some embodiments FGF-2 (also known as bFGF) is used at an effective concentration, in preferred embodiments FGF-2 is used at a concentration of 0.1ng/ml to 1 μ g/ml in more preferred embodiments FGF-2 is used at a concentration of 1ng/ml to 100ng/ml in more preferred embodiments FGF-2 is used at a concentration of 5ng/ml to 50ng/ml in most preferred embodiments FGF-2 is used at a concentration of 10ng/ml although the invention preferably uses FGF-2, the invention can also be performed using FGF-2 mimetics that have the same or similar signaling activity as FGF-2, characterized by binding to FGF-receptors to cause FGF-receptor-mediated signaling, wherein such activity is at least 10% of the signaling activity of FGF-2 to each FGF receptor.

In this culturing step C, the PSC is cultured in a cell culture medium suitable for the culture of the PSC. Examples of suitable cell culture media include TeSR-E8 base medium (Stemcell) and StemFlex medium (Gibco).

This culturing step is performed after step B disclosed above. The transition from step B to step C is generally characterized by the addition of the components of step C to an existing cell culture medium comprising PSCs and the matrix of step B. Step C is performed before step D, as described below. The conversion of step C to step D is characterized by a partial or complete exchange of the cell culture medium or, alternatively, the addition of the ingredients of step D to an existing cell culture medium.

In embodiments, step C is performed for a period of time from 6 hours to 4 days in preferred embodiments, step C is performed for a period of time from 12 hours to 3 days in a more preferred embodiment, step C is performed for a period of time from 1 day to 2 days in a most preferred embodiment, step C is performed for a period of time of 1 day.

Step C should begin on day-1 of the organoid production protocol. Depending on its duration, this step may be extended to day 0, 1, 2 or 3 of the protocol. In a most preferred embodiment, step C extends to day 0 of the regimen.

Step C is carried out in a suitable cell culture vessel. An exemplary suitable cell culture vessel is a 96-well plate with a U-shaped bottom and low adhesion properties. Typically, the cell culture vessel will not change when steps B and C disclosed herein are performed.

Step C is performed under conditions suitable for PSC survival. Exemplary suitable conditions include 37 ℃, 5% CO₂For example in a cell incubator.

Culturing the stem cell in a cell culture medium comprising retinoic acid and one or more inhibitors of SMAD signaling BENQ of PSC and matrix formation of step (C) to induce neurogenesis ("step D")

Culturing the formed BENO in a matrix according to the PSC composition treated in step C in a medium comprising retinoic acid and or more SMAD signaling pathway inhibitors, the treatment inducing neurogenesis from the PSC of step C and the matrix.

Retinoic acid is preferably used as a signaling activating molecule, all-trans retinoic acid [ (2E, 4E, 6E, 8E) -3, 7-dimethyl-9- (2,6, 6-trimethylcyclohexen-1-yl) non-2, 4,6, 8-tetraenoic acid ] is preferred, however, the invention can also be practiced using retinoic acid derivatives having the same or similar signaling activity as all-trans retinoic acid, which are typically characterized by binding to a retinoic acid receptor thereby causing retinoic acid receptor mediated signaling, wherein such activity is at least 10% of the signaling activity of all-trans retinoic acid, retinoic acid is used at effective concentrations in embodiments, retinoic acid concentrations ranging from 0.01 μ M to 100 μ M in preferred embodiments, retinoic acid concentrations ranging from 0.1 μ M to 10 μ M in more preferred embodiments, retinoic acid concentrations ranging from 0.5 μ M to 5 μ M in most preferred embodiments, retinoic acid concentrations ranging from 1 μ M.

For example, noggin may be used at a concentration of 0.1ng/ml to 1 μ g/ml, preferably 1ng/ml to 500ng/ml, more preferably 10ng/ml to 200ng/ml, most preferably 50ng/ml SB 432 may be used at a concentration of 0.1 μ M to 1mM, preferably 1 μ M to 100 μ M, more preferably 5 μ M to 50 μ M, most preferably 10 μ M using more than SMAD inhibitors may have a positive effect on the induction of neurogenesis (example 1. the use of more than SMAD inhibitors may consist of a combination of more preferably 15484 inhibitors of SMAD 1541. the preferred combination of SMAD inhibitors consists of a head protein of SMAD 431542 and a preferred combination of SMAD 431542 (SMAD) and a more preferred combination of SMAD inhibitors of SMAD 1542 and SMAD 43154431542 (example 1. the preferred combination of SMAD inhibitors consists of SMAD 431541. wt. 1. mu.5 μ M to 50 μ M, most preferred).

The formed BENO is cultured in any cell culture medium suitable for PSC culture. Exemplary cell culture media include Stemdiff neuron differentiation media (Stemcell), basal nerve cell media (Gibco), and the media used in example 2.

Step D of the disclosed method is typically performed after step C. The conversion of step C to step D is characterized by a partial or complete exchange of cell culture medium or, alternatively, the addition of the ingredients of step D to an existing cell culture medium. Step D is typically performed before step E, as described herein. The conversion of step D to step E is characterized by a partial or complete exchange of cell culture medium, or alternatively, the addition of the components of step E to an existing cell culture medium.

In embodiments, step D is performed for a period of 2 days to 16 days, in preferred embodiments, step D is performed for 4 days to 12 days, in a more preferred embodiment, step D is performed for 6 days to 10 days, in a most preferred embodiment, step D is performed for 8 days it has been found that it is advantageous to perform step D for 8 days (see example 8) compared to performing step D for 3 or 6 days, step D is performed for 10 days rather than for 8 days, and extending step D by does not significantly improve the results (see example 8). thus, in another particularly preferred embodiments, step D is performed for at least 8 days or 10 days.

The date this step is performed is "specified" by the duration of the previously performed steps B, C and D. typically, the start of step D is between day 0 and day 3. the end of step D is typically between day 2 and day 19. in the most preferred embodiment, step D extends from day 0 to day 8. in another particularly preferred embodiments, step D extends from day 0 to day 10.

Step D is performed using any suitable cell culture vessel exemplary suitable cell culture vessels include 96-well plates or 6-well plates with U-shaped bottoms and low adhesion or custom 3D printing or cast mold vessels for single or multiple organoid cultures.

Step D is performed under conditions suitable for survival of the formed BENO. Exemplary suitable conditions are 37 ℃, 5% CO₂For example in a cell incubator.

Culturing the formed BENO of step D in a cell culture medium comprising TGF- β and FGF-2 to enhance stromal cells Cellular and neurogenesis ("step E")

TGF- β treatment enhances the occurrence of stromal cells, while FGF-2 enhances neurogenesis, provided after step D is cultured in a medium comprising Transforming Growth Factor (TGF) β and fibroblast growth factor-2 (FGF-2).

TGF- β treatment enhances the development and function of stromal cells in emerging organoids, stromal cells are stromal cells of neural tissue exemplary stromal cells include glial cells TGF- β for use in the invention may be TGF- β, TGF- β, TGF- β, or mixtures thereof the invention may also be performed using a TGF- β mimic with the same or similar signaling activity as TGF- β 4, characterized by binding to TGF- β receptors resulting in TGF- β 7-receptor mediated signaling, where such activity is at least 10% of TGF- β signaling activity, preferably TGF- β for use in the invention is TGF- β. TGF- β is used at effective concentrations in β 3 embodiments TGF- β is at a concentration of 0.1ng/ml to 100ng/ml in preferred embodiments TGF- β is at a concentration of 0.3ng/ml to 30ng/ml in preferred embodiments, TGF-5 ng/ml is at a concentration of 0.1ng/ml to 30ng/ml in more preferred embodiments TGF- β ml to 10 ng/ml.

In , FGF-2 (also known as bFGF) is used at an effective concentration in the range of 0.1ng/ml to 1 μ g/ml in preferred embodiments FGF-2 is used at a concentration of 1ng/ml to 100ng/ml in more preferred embodiments FGF-2 is used at a concentration of 5ng/ml to 50ng/ml in most preferred embodiments FGF-2 is used at a concentration of 10ng/ml although the invention is preferably practiced with FGF-2, the invention can also be practiced with FGF-2 mimetics that have the same or similar signaling activity as FGF-2, characterized by binding to FGF-receptors and thereby causing FGF-receptor-mediated signaling, wherein such activity is at least 10% of the signaling activity of FGF-2 to each receptor.

Exemplary cell culture media include Stemdiff neuronal differentiation media (Stemcell), basal neural cell media (Gibco), and the media used in example 2.

Step E is typically performed after step D. The conversion of step D to step E is characterized by a partial or complete exchange of cell culture medium or, alternatively, the addition of the ingredients of step E to an existing cell culture medium. Typically, step E is performed before step F, as described below. The conversion of step E to step F is characterized by a partial or complete exchange of cell culture medium, or alternatively, the addition of the ingredients of step F to an existing cell culture medium.

In some embodiments of , step E is performed for a period of 2 days to 16 days, in preferred embodiments step E is performed for 4 days to 12 days, in more preferred embodiments step E is performed for 6 days to 10 days, in most preferred embodiments step E is performed for 7 days it has been found that there is no significant disadvantage in performing step E for 5 days as compared to performing 7 days, and conversely, performing step E for 2 days results in poor BENO aggregation (see example 8). thus, in another particularly preferred embodiments, step E is performed for up to 7 days or 5 days.

The date on which this step is performed is "specified" by the duration of the previously performed steps B, C, D and E. typically, the start of step E is from day 3 to day 15, the end of step E is from day 5 to day 20. in the most preferred embodiment, step E is extended from day 8 to day 15. in another particularly preferred embodiments, step E is performed from day 10 to day 15.

Step E is performed using any suitable cell culture vessel. Exemplary suitable cell culture vessels include 6-well or custom 3D printed or molded vessels for single or multiple organoid cultures. The cell culture vessel is generally not altered between steps D and E. Step E may be performed by changing the cell culture vessel.

Step E is carried out under conditions suitable for survival of the formed BENO. Exemplary suitable conditions are 37 ℃, 5% CO₂For example in a cell incubator.

Culturing of step E in cell culture media comprising TGF- β and or more notch signaling inhibitors BENO formed to enhance stromal cell genesis and neural differentiation ("step F").

Treatment of the formed BENO provided after culturing step E with TGF- β in culture media containing transforming growth factor β (TGF- β) and or more notch signaling inhibitors enhances the occurrence of stromal cells while inhibiting notch signaling enhances neural differentiation.

TGF- β used in the present invention may be TGF- β, TGF- β, TGF- β, or mixtures thereof, the present invention may also be performed using a TGF- β 5 mimetic having the same or similar signaling activity as TGF- β 4, characterized by binding to a TGF- β receptor causing TGF- β -receptor mediated signaling, wherein such activity is at least 10% of TGF- β signaling activity TGF- β is used at effective concentrations.

Suitable Notch signaling inhibitors include N- [ (3, 5-difluorophenyl) acetyl ] -L-alanyl-2-phenyl ] glycine-1, 1-dimethylethyl ester (DAPT), compound e (stem cell technologies), and gamma-secretase inhibitors such as those described in olswaskas-Kuprys et al, oncotargets and therapyp, 2013. the Notch signaling inhibitors are used in effective concentrations.

Exemplary cell culture media include Stemdiff neuronal differentiation media (Stemcell), basal neuronal cell media (Gibco) and the media used in example 2.

Step F is generally carried out after step E. The conversion of step E to step F is characterized by a partial or complete exchange of cell culture medium, or alternatively, the addition of the components of step F to an existing cell culture medium.

In embodiments, step F is performed for a period of time from 5 days to 95 days, in preferred embodiments step F is performed for a period of time from 7 days to 50 days, in more preferred embodiments step F is performed for a period of time from 10 days to 20 days, and in most preferred embodiments step F is performed for a period of time of 13 days.

The date on which this step is performed "assignment" depends on the duration of the previously performed steps B, C, D, E and F. Typically, the starting point for step F is between day 4 and day 35. The end of step F is between day 9 and day 100. In a most preferred embodiment, step F extends from day 15 to day 28.

Step F is performed using any suitable cell culture vessel exemplary suitable cell culture vessels are 6-well plates or custom 3D printing or cast mold vessels for single or multiple organoid culture.

Step F is performed under conditions suitable for survival of the formed BENO. Exemplary suitable conditions are 37 ℃, 5% CO₂For example in a cell incubator.

Completion of step F of the methods disclosed herein provides that after step F, additional cultures may be further -step cultures or expanded BENO under appropriate conditions may lead to the development of other characteristics of BENO and/or optimized neurogenesis, depending on the desired application of the BENO.

Characteristics and advantages of the neuronal organoids produced according to the method of the invention.

The neuronal organoids (i.e., BENO) produced by the methods disclosed herein exhibit cortical development, neurogenesis, and neuroglialization. BENO includes neuronal cells and stromal cells. Stromal cells (e.g., glial cells) are important for providing an environment that promotes neurogenesis that occurs when practicing the disclosed methods. The multicellular complexity of the human brain is effectively reconstructed by the simultaneous neurogenesis and neuroglialization of defined growth factors and small molecules disclosed herein.

For example, BENO described herein forms a functional neuronal network characterized by neuronal function, including the formation of functional synapses, the formation of hierarchical networks, GABAergic networks, and the synchronization of neurons (example 5). these neuronal functions represent at least important advantages over conventional neuronal organoids.

Moreover, the BENO produced by the process of the invention is preferably produced under well-defined conditions (e.g.serum-free). This means that the BENO can be reproduced reliably, since changes originating from undefined or undefined chemical compositions are eliminated.

Application of BENO

The BENO produced by the method of the present invention can be used for so-called phenotypic drug screening. Unlike target-specific drug screening techniques, phenotypic drug screening is not focused on binding of candidate molecules to specific targets, but on the effect of target molecules on the phenotype. A prerequisite for such phenotypic drug screening is the presence of an appropriate model that can mimic the phenotype of the disease under study. When studying various diseases associated with neural tissue, the BENO of the present disclosure may provide a model of such diseases. Diseases for which the present invention may provide suitable drug screening models include stroke, encephalitis diseases, neurodegenerative diseases (e.g., parkinson's disease, alzheimer's disease, as in example 6), neuroinflammatory diseases (e.g., multiple sclerosis), traumatic injuries (e.g., injuries resulting from brain surgery), ion channel diseases (e.g., epilepsy), and psychiatric diseases (including autism and schizophrenia, as in example 7).

BENO can be used to discover and refine drugs by phenotypic drug screening. Such uses of BENO include the discovery and refinement of drugs that induce or enhance the repair, regeneration, protection and prevention of disease of brain and neural tissue.

PSCs useful for such a BENO model can be obtained from healthy individuals or diseased patients. Alternatively, gene editing of pluripotent stem cells can be applied to create any genetic and epigenetic modification of interest. Thus, after the production of the BENO composition from PSCs according to the methods of the present disclosure, BENO allows phenotypic tissue screening with high predictive value. The use of BENO according to the present invention offers many advantages over conventional models due to the higher degree of maturation of neural tissue, cellular complexity and hierarchical network function. Furthermore, the simplicity of the disclosed method for the production of BENO makes it easy to achieve high throughput phenotypic screening. Disease simulation (e.g., hypoxia-induced stroke-like injury) can be performed on health and disease modeled BENO. Phenotypic readings include effects on tissue formation, electrical connections, cell death and cell proliferation of specific cell types in BENO. Phenotypic drug screening allows the definition and validation of various drug targets. This may therefore provide a basis for subsequent compound screening to identify compounds having, for example, regenerative, reparative, disease-altering or protective biological activity.

BENO produced according to the methods of the present disclosure may also be used in drug safety screens to test, for example, the potential of a substance to induce electrical interference (seizures), degeneration, cell death, or other cellular abnormalities in neural tissue.

The BENO produced by the method of the invention may also be used in studies involving the mode of action of drugs, for example in preclinical trials conducted in parallel with clinical trials.

The BENO produced by the method of the present invention may also be used for personalized medical purposes. For example, patient-derived ipscs can be used to model disease and test personalized therapies. The BENO of the present invention is particularly useful for testing therapies for diseases associated with neural tissue, such as neurodegenerative or neuroinflammatory diseases. All possible therapy options can be explored and tested using the described BENO, for example, therapy by drugs, biologics (such as antibodies or non-coding RNAs), gene editing, or combinations thereof.

In some embodiments co-culturing with EHM (engineered myocardium) and BSM (bioengineered skeletal muscle) may be used to study the development of neuromuscular junctions (junctions). particularly, co-culturing of BENO with EHM may be used to study the interaction of neurons and pacemakers or neurons and cardiomyocytes.

BENO of the present invention may also provide regenerated tissue for scientific or therapeutic purposes, for example, BENO may be damaged to study repair and regeneration following pharmacological or biophysical (e.g., electro-conditioning) treatments, or a BENO with specific brain functions (e.g., dopamine production and release to combat Parkinson's disease) may be constructed further to link the BENO to organs or to serve as a machine-organ interface to achieve control of debilitated (enervated) organs (e.g., control of skeletal muscles).

Reagent kit

In embodiments, the kit comprises a PSC, a matrix, a suitable medium, and a desired supplement (ROCKi, FGF-2, retinoic acid, or more inhibitors of SMAD signaling, TGF- and or more inhibitors of notch signaling). in other embodiments, the kit comprises a matrix, a suitable medium, and a desired supplement (ROCKi, FGF-2, retinoic acid, or more inhibitors of SMAD 387 signaling, TGF-585, and or more inhibitors of notch signaling). in other embodiments, the kit comprises a matrix and a desired supplement (ROCKi, FGF-2, retinoic acid, or more inhibitors of SMAD signaling, TGF- β, and or more inhibitors of notch signaling). in other embodiments, the kit comprises a matrix and at least 4 desired supplements (ROCKi, FGF-2, ki- or more inhibitors of SMAD signaling, TGF- β, and or more inhibitors of notch signaling β).