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Definitions

• PSCs pluripotent stem cells
• NPCs neural progenitor cells
• Another drawback associated with known methods is a lack of neuronal network function of the produced organoids, which thus significantly limits any investigation into neuronal functionality and plasticity.
• Disease modeling and drug development is also restricted due to the remote phenotypic resemblance of known produced neuronal organoid structures compared to normal brain tissue.
• the present invention provides methods permitting both robust and reproducible neural differentiation in a well-defined 3D cell culture system, which further provides a sound basis for investigations into the formation, and plasticity characteristics, of a functional neuronal network.
• the organoids of the present invention further offer a valuable tool in drug development.
• such organoids can meaningfully decrease the typically exorbitant costs associated with both preclinical and clinical drug development, at least because of a decreased need for animal-based experimentation and a reduced number of patients required in clinical trials when predicting post marketing outcome.
• novel biologicals e.g., non- coding RNA therapeutics
• genome editing e.g., using CRISPR-based platforms
• the present invention relates to a method for producing a bioengineered neuronal organoid (BENO) from pluripotent stem cells (PSCs), the method comprising:
• step (B) culturing the PSCs of step (A), embedded in a matrix immersed in cell culture medium;
• step (C) culturing the PSCs in said matrix of step (B) in cell culture medium comprising a Rho-associated kinase inhibitor (ROCKi) and FGF-2;
• ROCKi Rho-associated kinase inhibitor
• step (D) culturing the forming BENO originating from PSCs and matrix of step (C) in cell culture medium comprising retinoic acid and one or more inhibitors of SMAD signaling to induce neurogenesis;
• step (E) culturing the forming BENO of step (D) in cell culture medium comprising TGF-beta and FGF-2 to enhance genesis of stromal cells and neurogenesis;
• step (F) culturing the forming BENO of step (E) in cell culture medium comprising TGF-beta and one or more inhibitors of notch signaling to enhance genesis of stromal cells and neurodifferentiation.
• the matrix does not comprise Matrigel. In other embodiments, the matrix does not comprise Matrigel or other components of natural origin having a non- defined composition. 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.
• the BENO is produced within a 3D environment, preferably wherein the 3D environment is defined by the matrix.
• the stromal cells comprise glial cells.
• the medium of step (D) comprises at least two inhibitors of SMAD signaling, preferably wherein the inhibitors of SMAD signaling comprise noggin and SB 431542. In some embodiments, the inhibitors of SMAD signaling are noggin and SB 431542.
• the inhibitor of notch signaling of step F is DAPT.
• the inhibitors of SMAD signaling are noggin and SB 431542 and the inhibitor of notch signaling of step F is DAPT.
• the matrix is collagen employed at a concentration between 0.05 mg/ml and 50 mg/ml, preferably between 0.1 mg/ml and 10 mg/ml, more preferably between 0.5 mg/ ml and 5 mg/ ml, most preferably at a concentration of 1 mg/ ml.
• retinoic acid is employed at an effective concentration between 0.01 ⁇ and 100 ⁇ , preferably between 0.1 ⁇ and 10 ⁇ , more preferably between 0.5 ⁇ and 5 ⁇ , most preferably at a concentration of 1 ⁇ .
• noggin is employed at an effective concentration of 0.1 ng/ml - 1 ⁇ g/ml, preferably 1 ng/ml - 500 ng/ml, more preferably 10 ng/ml— 200 ng/ml, most preferably 50 ng/ml.
• SB 431542 is employed at an effective concentration of 0.1 ⁇ - 1 mM, preferably 1 ⁇ - 100 ⁇ , more preferably between 5 ⁇ and 50 ⁇ , most preferably 10 ⁇ .
• TGF- beta is employed at an effective concentration of between 0.1 ng/ml and 100 ng/ml, preferably between 0.3 ng/ml and 30 ng/ml, more preferably between 1 ng/ml and 10 ng/ml, most preferably at a concentration of 5 ng/ml.
• FGF-2 is employed at an effective concentration of 0.1 ng/ml and 1 ⁇ g/ml, preferably between 1 ng/ml and 100 ng/ml, more preferably between 5 ng/ml and 50 ng/ml, most preferably at a concentration of 10 ng/ml.
• DAPT is employed at an effective concentration of between 0.01 ⁇ and 100 ⁇ , preferably between 0.1 ⁇ and 10 ⁇ , more preferably between 0.5 ⁇ and 5 ⁇ , most preferably at a concentration of 2.5 ⁇ .
• retinoic acid is employed at a concentration of between 0.5 ⁇ and 5 ⁇
• noggin is employed at a concentration of 10 ng/ml - 200 ng/ml
• SB 431542 is employed at a concentration of between 5 ⁇ and 50 ⁇
• TGF-beta is employed at a concentration of between 1 ng/ml and 10 ng/ml
• FGF-2 is employed at a concentration of between 5 ng/ml and 50 ng/ml
• DAPT is employed at a concentration of between 0.5 ⁇ and 5 ⁇ .
• retinoic acid is employed at a concentration of between 0.5 ⁇ and 5 ⁇
• noggin is employed at a concentration of 10 ng/ml - 200 ng/ml
• SB 431542 is employed at a concentration of between 5 ⁇ and 50 ⁇
• TGF-beta is employed at a concentration of between 1 ng/ml and 10 ng/ml
• FGF-2 is employed at a concentration of between 5 ng/ml and 50 ng/ml
• DAPT is employed at a concentration of between 0.5 ⁇ and 5 ⁇
• the matrix is collagen employed at a concentration between 0.1 mg/ml and 10 mg/ml.
• retinoic acid is employed at a concentration of 1 ⁇
• noggin is employed at a concentration of 50 ng/ml
• SB 431542 is employed at a concentration of 10 ⁇
• TGF-beta is employed at a concentration of 5 ng/ ml
• FGF-2 is employed at a concentration of 10 ng/ml
• DAPT is employed at a concentration of 2.5 ⁇ .
• retinoic acid is employed at a concentration of 1 ⁇
• noggin is employed at a concentration of 50 ng/ml
• SB 431542 is employed at a concentration of 10 ⁇
• TGF-beta is employed at a concentration of 5 ng/ ml
• FGF-2 is employed at a concentration of 10 ng/ml
• DAPT is employed at a concentration of 2.5 ⁇
• the matrix is collagen employed at a concentration between 0.5 mg/ml and 5 mg/ml
• the PSCs are animal cells. In some embodiment, the PSCs are mammal cells. In some embodiment, the PSCs are rodent (e.g., mouse or rat) or human cells. In preferred embodiments, the PSCs are human PSCs.
• the PSCs of the present invention are not produced using a process involving a modification to the germ line genetic identity of a human being or involving a use of a human embryo for industrial or commercial purposes.
• step (A) and step (B) are performed on day -1.
• step (C) is performed from day -1 to day 0.
• step (D) is performed from day 0 to day 8.
• step (E) is performed from day 8 to day 15.
• step (F) is performed from day 15 to at least day 28.
• step (A) and step (B) are performed on day -1 and step (C) is performed from day -1 to day 0.
• step (A) and step (B) are performed on day -1
• step (C) is performed from day -1 to day 0
• step (D) is performed from day 0 to day 8.
• step (A) and step (B) are performed on day -1, step (C) is performed from day -1 to day 0, step (D) is performed from day 0 to day 8, and step (E) is performed from day 8 to day 15.
• step (A) and step (B) are performed on day -1, step (C) is performed from day -1 to day 0, step (D) is performed from day 0 to day 8, step (E) is performed from day 8 to day 15 and step (F) is performed from day 15 to at least day 28.
• step (A) and step (B) are performed on day -1
• step (C) is performed from day -1 to day 1
• step (D) is performed from day 0 to day 10
• step (E) is performed from day 10 to day 15
• step (F) is performed from day 15 to at least day 28.
• the invention provides neuronal organoids, e.g., bioengineered neuronal organoids (BENOs), characterized in that the neuronal cells of the neuronal organoid are organized in a functional neuronal network.
• the invention provides bioengineered neuronal organoids (BENOs) produced by the method of the invention.
• the invention is directed at the use of BENOs produced by methods of the invention as model for a disease.
• the invention is directed at the use of BENOs produced by methods of the invention as models for a disease related to neural tissue.
• the invention is directed at the use of BENOs produced by methods of the invention as models for a disease selected from the group consisting of stroke, brain inflammation disorders, neurodegenerative diseases, neuroinflammatory diseases, traumatic injury, channelopathy, and psychiatric diseases.
• the invention is directed at the use of BENOs produced by methods of the invention as models for a disease selected from the group consisting of neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease), neuroinflammatory diseases (e.g., multiple sclerosis), traumatic injury (e.g., brain- surgery-induced injury), channelopathy (e.g., epilepsy) and psychiatric diseases (e.g., autism, schizophrenia).
• a disease selected from the group consisting of neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease), neuroinflammatory diseases (e.g., multiple sclerosis), traumatic injury (e.g., brain- surgery-induced injury), channelopathy (e.g., epilepsy) and psychiatric diseases (e.g., autism, schizophrenia).
• neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease
• neuroinflammatory diseases e.g., multiple sclerosis
• traumatic injury e.g., brain- surgery-induced injury
• channelopathy e.g., epi
• the invention is directed at the use of BENOs produced by methods of the invention as model for a disease, wherein the BENOs are in co-culture with another tissue engineering platform.
• the other tissue engineering platform is selected from the group consisting of EHM (Engineered Heart Muscle), BSMs (bioengineered skeletal muscle), ESM (Engineered Skeletal Muscle), ELT (Engineered Liver Tissue), and ECT (Engineered Connective Tissue).
• the other tissue engineering platform is selected from the group consisting of tumor models (e.g., tumor brain invasion, metastases spread) and leukocyte infiltration models (e.g., autoimmue disease).
• the invention is directed at the use of BENOs produced by methods of the invention in drug screening, such as drug discovery and drug refinement by phenotypic drug screening.
• This use of BENOs includes, but is not limited to the discovery and refinement of drugs that may induce or enhance repair, regeneration, protection, and disease prevention in brain and neural tissue.
• the invention is directed at a kit for practicing a method of the invention.
• the kit contains PSCs, a matrix, suitable media and the required supplements (ROCKi, FGF-2, retinoic acid, one or more inhibitors of SMAD signaling, TGF- beta, and one or more inhibitors of notch signaling).
• a kit contains a matrix, suitable media and the required supplements (ROCKi, FGF-2, retinoic acid, one or more inhibitors of SMAD signaling, TGF-beta, and one or more inhibitors of notch signaling).
• a kit contains a matrix and the required supplements (ROCKi, FGF-2, retinoic acid, one or more inhibitors of SMAD signaling, TGF-beta, and one or more inhibitors of notch signaling). In other embodiments, a kit contains a matrix and at least 4 of the required supplements (ROCKi, FGF-2, retinoic acid, one or more inhibitors of SMAD signaling, TGF- beta, and one or more inhibitors of notch signaling).
• FIG. 1 Scheme of typical neural differentiation from stem cells.
• FIG. 2 Enhanced neurogenesis under dual SMAD signaling pathway inhibition.
• FIG. 2A IF analysis of tissues treated exclusively with noggin or noggin and SB431542. Neurons were visualized using an antibody against neurofilament (FITC-green) and nuclei with DAPI (Blue). Bar graph represents 10 ⁇ . Quantification of mean fluorescence ratio from whole tissues is presented in the right-hand graph.
• FIG. 3 FGF-2 enhances neuronal differentiation.
• FIG. 3 A Increasing input of pluripotent stem cells resulted in enhanced neurofilament staining at BENO culture day 28.
• FIG. 3B Proliferative effect of FGF-2 on stem cells and NPCs. The amount of neurons as well as network complexity inside the tissues as visualized with neurofilament staining is higher in tissues treated with FGF-2 at days 8-15.
• FIG. 4 Neuronal transcript analysis upon BDNF and GDNF treatment. Neither PAX6 nor MAP2 expression was enhanced upon the addition of BDNF and GDNF in culture from day 10 to day 28. BENOs were analyzed at day 28.
• FIG. 5 Enhanced neurogenesis by notch inhibition. DAPT treatment from day 15 to day 28 increased the abundance of PAX6 transcripts, thus suggesting a higher amount of neuronal commitment.
• FIG. 6 Evaluation of neurogenic and gliogenic potential of different protocols.
• FIG. 6A Summarizes a scheme of treatment performed in the individual protocols of Example 1.
• FIG. 6B Transcriptomic time course analysis of BENO generation. OCT4 was used as stem cell marker, GFAP as a glia marker, PAX6 as NPC and neuron marker and finally MAP2, GRIN1 and GABBR2 as mature neuron marker. Data were normalized to GAPDH.
• FIG. 6C Whole mount IF analysis of BENOs at day 60. Neurofilament, MAP2, synaptophysin and GFAP were used to stain neurons, mature neurons, synapsis and glia respectively.
• FIG. 6D BENO activity calculated by the detection of calcium activity documented in 5 areas of the tissue.
• FIG. 7 Heat maps from RNAseq analysis during the time course of development displaying RNAseq data on neurogenesis and maturation in BENOs.
• FIG. 7A Markers depicting different state of differentiation of stem cells into neurons and glia.
• FIG. 7B Markers of different neuronal identities.
• FIG. 7C Cortical layer markers.
• FIG. 7D Different maturation proteins as receptors, ion channels, and synapsis related proteins. Performed in collaboration with Dr. Rashi Haider/Prof. A. Fischer (DZNE).
• FIG. 8 Cortical layer development in BENOs. TBR2 + subventricular zone progenitors are concentrically migrating from the middle of the organoid to the periphery. There, CTIP2 marks the deep layer neurons.
• FIG. 10 Neuronal network function in BENOs suggests integrated and hierarchical synaptic functions. Fura-4 stained neurons are shown at the left panel. Matlab analysis of different regions of interest (ROI) indicated 12 different traces shown in the right panel. Before GABAR inhibition ROI (2,3), ROI (4,5), ROI (6, 7) and ROI (11,12) were synchronized. GABAR inhibition led to cell desynchronization and upon 10 min washout the cells synchronized again. Performed in collaboration with Dr. Guobin Bao/Prof. D. Schild (UMG).
• FIG. 11 A qPCR analysis of relative PAX6 transcript expression 15 days upon BENO generation using the protocols indicated in the scheme.
• FIG. 11B Defining the maximum duration of BENO treatment with NCM (step D). qPCR analysis of relative PAX6 transcript expression 15 days upon BENO generation using the protocols indicated in the scheme.
• FIG. 11C Immunofluorescence analysis of PAX6/ki67-positive cells to mark proliferating neuronal progenitor cells.
• organoid refers to a tissue culture forming a three- dimensional assembly, which mimics, at least partially, the structure and/ or function of an organ, such as a human organ.
• Organoids can be generated from pluripotent stem cells in, for example, a three-dimensional (3D) environment.
• 3D environment for organoids is a spheroid- shaped 3D environment.
• An organoid may further be regarded as a miniaturized and simplified version of an organ.
• BENO biologically engineered neuronal organoid
• a BENO may be regarded as a miniaturized and simplified model of a neural organ, including the brain, or of neural tissue existing within an organ or controlling an organ, for instance, neural tissue present within the heart (e.g., sympathetic nervous system) and skeletal muscle (e.g., nicotinergic nerve endings at skeletal neuromuscular junctions).
• a "forming BENO” as used herein is a composition of cells and matrix that is in the process of developing into a BENO.
• a forming BENO is characterized in that its cellular and matrix material has been subjected to step C of the methods of the invention, but either has not yet been subjected to step F of the methods of the invention or step F is not yet finished.
• 3D environment represents a structure extending in all three dimensions of space.
• a 3D environment corresponds to a structure wherein cells are arranged in a three dimensional space in relation to each other.
• One example of a 3D environment is a spherical arrangement.
• Different from a 3D environment is a 2D environment, wherein the cells are arranged in a single layer, for example there is no difference in one of the dimensions of the spatial relationship between the cells.
• 3 D cell culture system refers to cell culturing in a 3 D environment, at least initially defined by a 3D matrix.
• pluripotent stem cells are cells having the property of self-replication, and a propensity to differentiate into cells found in the three germ layers (endoderm, ectoderm and mesoderm). PSCs exist in an undifferentiated state and are characterized by abundant expression of sternness factors such as Oct-3/4, SSEA-4, and TRA1- 60, a property of self-replication, and a propensity to differentiate into cells of the three germ layers (endoderm, ectoderm and mesoderm).
• sternness factors such as Oct-3/4, SSEA-4, and TRA1- 60
• PSCs can be derived from numerous sources, including but not limited to, induced pluripotent stem cells (iPSCs), parthenogenetic stem cells, stem cells generated by nucleus transfer and embryonic stem cells (ESCs) and combinations thereof.
• ESCs can be from existing ESC lines.
• the PSCs of the present invention are not produced using a process involving a modification to the germ line genetic identity of a human being or involving a use of a human embryo for industrial or commercial purposes.
• PSCs further include PSC cell lines.
• the origin of PSCs as used herein is not particularly limited, but mammal- derived cells are preferred, more preferably the cells are of human origin.
• the term ' ' r epr ogramming' ' refers to methods wherein a more specialized cell or a cell in some other form of advanced stage of development can be converted into a pluripotent cell.
• differentiated cell refers to a cell that has developed from an unspecialized precursor phenotype to a specialized phenotype.
• an embryonic cell can differentiate into an epithelial cell lining of the intestine.
• differentiated cells can be isolated from a fetus or from a live animal.
• Induced pluripotent stem cells represent a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell. Induced pluripotent stem cells are considered to be similar, if not identical to, natural pluripotent stem cells, including embryonic stem cells, for instance, in terms of the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability.
• NPCs neural progenitor cells
• the progeny of NPCs can be either neuronal cells (such as neuronal precursors or mature neurons) or glial cells (such as glial precursors, mature astrocytes, or mature oligodendrocytes).
• NPCs do not normally produce progeny of other embryonic germ layers when cultured by themselves in vitro unless, for example, they are dedifferentiated or reprogrammed in some fashion. Different from stem cells like PSCs, NPCs have limited proliferative ability and thus do not exhibit an ability of self-maintenance.
• Matrigel is a composition derived from Engelbreth-Holm- Swarm mouse sarcomas (Kleinman et ah, Biochemistry, 1982). Matrigel is a mixture that is not precisely defined chemically, but generally comprises laminin, collagen IV, heparin sulfate proteoglycans, entactin, and growth factors. Matrigel is commonly used as a matrix for cell cultivation.
• compositions for which the chemical composition is known in a sufficient degree of accuracy For example, at most 10 %, preferably at most 5%, of the total content of the composition is chemically uncharacterized or varying in different samples of the composition.
• Components of natural origin having a non-defined composition as used herein relates to compositions that have been isolated from natural sources (cells or tissues from animals, plants, fungi, and protists cells or viruses) that are chemically not precisely defined. For example, at least 10 % of the total content of the composition must be uncharacterized or varying in different samples of the composition.
• components of natural origin having a "non-defined composition” include sera and Matrigel.
• FGF-2 Fibroblast growth factor-2
• FGF-2 Fibroblast growth factor-2
• bFGF basic fibroblast growth factor
• a "neuronal network” as used herein represents a group of one or more interconnected neurons.
• the connection between the neurons in a neuronal network permits a transmission of information from one neuron to the other (s).
• the connection between neurons can be via synapses.
• the presence of a neuronal network can be readily confirmed, for example, through the use calcium imaging of the neurons.
• Neuronal network organization refers to the organization of a group of neurons as a neuronal network. This organization of neurons as a neuronal network can create a hierarchical network.
• a "functional neuronal network” as used herein refers to a neuronal network, which displays a transmission of electrochemical information from one neuron to the other. Functional neuronal networks require the formation of functional synapses. Functional neuronal networks are characterized by activity patterns of the network which may include synchronized electrical activity of more than one neuron of the network or patterns showing functional interdependencies between neurons of the network, including neuron activation or inhibition patterns. The presence of a neuronal network can be confirmed, for example, by calcium imaging of the neurons. In particular, the reaction of a group of neurons to signaling molecules or inhibition of receptors of signaling molecules (e.g., GABA receptors) can confirm the presence of a functional neuronal network.
• signaling molecules or inhibition of receptors of signaling molecules e.g., GABA receptors
• One example evidencing a functional neuronal network is where the neurons in the network demonstrate synchronized calcium signals (e.g., calcium spikes), which become un-synchronized following the addition of a receptor inhibitor that inhibits a neuronal signaling molecule and subsequently re-synchronize upon inhibitor removal.
• synchronized calcium signals e.g., calcium spikes
• Neuronal network function as used herein is the transmission of electrochemical information from one neuron to the other.
• inducing means initiating and/or enhancing a particular physiological effect such as cell proliferation or cell differentiation.
• Neurogenesis refers to the differentiation and/ or proliferation of cells towards fully differentiated neural cells which are not able to differentiate further.
• neurogenesis includes the differentiation of PSCs to NPCs, the proliferation of NPCs, the differentiation of NPCs into more differentiated neural cells, for instance, either neuronal cells (such as neuronal precursors or mature neurons) or glial cells (such as glial precursors, mature astrocytes, or mature oligodendrocytes), or the proliferation of more differentiated neural cells.
• neurodifferentiation means the differentiation of neural cells towards fully differentiated neural cells which are not able to differentiate further.
• neurodifferentiation includes the differentiation of NPCs into more differentiated neural cells, for instance, either neuronal cells (such as neuronal precursors or mature neurons) or glial cells (such as glial precursors, mature astrocytes, or mature oligodendrocytes), or the differentiation of more differentiated neural cells into even more differentiated neural cells, for instance, from neuronal precursors to mature neurons.
• Embedding of cells in a matrix refers to the interaction of cells with, and/ or the attachment of cells to, a matrix. This process is regulated by cell-matrix interaction, for instance, through cellular receptors including integrins.
• matrix means a material that can create a 3D environment suitable for embedding cells.
• the matrix of the instant invention forms a hydrogel structure.
• suitable matrices include collagen or synthetic collagen mimics.
• Hydrogel refers to a network of hydrophilic polymers comprising water, but not being water-soluble. Hydrogel molecules are chemically and/or physically connected, for example, by covalent or ionic bonds or entanglement to thereby form a 3D environment. A hydrogel network can also be a natural or synthetic polymeric network.
• stromal cells refers to neural cells that are not neuronal cells.
• exemplary stromal cells include glial cells, such as glial precursors, mature astrocytes, or mature oligodendrocytes.
• signaling refers to the transmission of information (signals) within a cell or between two or more cells.
• the signaling transmission can occur by chemical reaction means (such as phosphorylation, protein cleavage), through a release of signaling molecules (including ions, neurotransmitter), or alternatively, through changes in the immediate electrochemical potential.
• tissue engineering platform as used herein means an in-vitro assembly of cells that is designed to mimic structural and/or functional features of tissue, preferably human tissue.
• phenotypic drug screening refers to screening of the applicability of new or existing drugs based on their effect on a phenotype of a model system.
• bioengineering and “bioengineered” as used herein refers to methods to manipulate biological systems and biological material. Examples for bioengineering are molecular cloning, transfection, transduction, and influencing cells using chemicals or other materials.
• disease modeling refers to the process of generating a model for a disease that mimics some or all features of the disease, at least partially.
• Diseases models can be used to assess the influence of compounds such as drugs on the diseases or some features of the disease.
• Exemplary disease models are cell cultures, organoids and mouse models.
• the present invention provides methods for producing bioengineered neuronal organoids (BENOs) under chemically defined conditions, which are reproducible and result in a consistent product.
• Staged SMAD and notch inhibition in the presence of retinoic acid and FGF- 2 for enhanced neurogenesis with TGF-beta for supporting gliogenesis represents a unique combination of biological activities for neurogenesis in a matrix environment, resulting in the formation of functional neuronal networks.
• One exemplary suitable matrix of the invention is a collagen hydrogel.
• neuro- and gliogenesis could be controlled using defined factors in a matrix environment that is not itself inducing neurogenesis; in contrast to, for example, a Matrigel matrix, which is known to induce neurogenesis since its introduction by Kleinman and colleagues in the 1990's (e.g., Jucker et a/., J Neurosd Res. 1991)
• the methods of the present invention were developed through several iterations, for instance, as disclosed by Example 1, resulting in structures displaying pronounced neuronal network organization and function, which is supported by co-developing stroma cells such as glia.
• the network organization and function for example, the formation of functional synapses between different neuronal cells (and thus, a functional hierarchy as described in Example 5) of the neuronal organoids disclosed herein offer a number of advantages over conventional organoid structures.
• the neuronal organoids disclosed herein can be produced show high consistency due to defined culturing conditions, comprise functional neuronal networks and can be produced in short time frames (e.g., 29 days from mixing of PSCs and matrix).
• the presently disclosed methods of the invention comprise a number of steps for implementation. These steps are: (A) providing a source of PSCs;
• step (B) culturing the PSCs of step (A), embedded in a matrix immersed in cell culture medium;
• step (C) culturing the PSCs in said matrix of step (B) in cell culture medium comprising a Rho-associated kinase inhibitor (ROCKi) and FGF-2;
• ROCKi Rho-associated kinase inhibitor
• step (D) culturing the forming BENO originating from PSCs and matrix of step (C) in cell culture medium comprising retinoic acid and one or more inhibitors of SMAD signaling to induce neurogenesis;
• step (E) culturing the forming BENO of step (D) in cell culture medium comprising TGF-beta and FGF-2 to enhance genesis of stromal cells and neurogenesis;
• step (F) culturing the forming BENO of step (E) in cell culture medium comprising TGF-beta and one or more inhibitors of notch signaling to enhance genesis of stromal cells and neurodifferentiation.
• PSCs pluripotent stem cells
• the present invention relates to producing bioengineered neuronal organoids (BENOs) from pluripotent stem cells (PSCs).
• Pluripotent stem cells can be obtained from a variety of sources, including but not limited to, induced pluripotent stem cells (iPSCs) (which can be generated by a reprogramming of cell types including fibroblasts, keratinocytes, bone marrow derived cells or blood derived cells such as cord blood derived cells), parthenogenetic stem cells, stem cells generated by nucleus transfer, and embryonic stem cells and/ or mixtures thereof.
• iPSCs induced pluripotent stem cells
• the PSCs of the present invention are not produced in a process that involves modifying the germ line genetic identity of a human being or which involves the use of a human embryo for industrial or commercial purposes.
• the methods of the invention can also be performed using PSC cell lines, for example, the iPSC-Gl cell line described in Tibury et al., Circulation, 2017.
• PSCs are characterized by an abundant expression of sternness factors such as Oct-3/4, SSEA-4, and TRAl-60 in an undifferentiated state, a property of self-replication, and a propensity to differentiate into cells of the three germ layers (endoderm, ectoderm and mesoderm).
• the PSCs can also be induced PSCs (iPSCs).
• PSCs Prior to use in the presently claimed methods, PSCs are cultured under appropriate conditions known in the art. PSCs may be, as necessary, cultured according to a standard maintenance procedure, e.g., growth on a maintenance support such as Matrigel. PSCs are grown in any appropriate cell culture medium known in the art.
• An exemplary cell culture medium is TeSR-E8 Basal medium (Stemcell), optionally comprising Rho-associated protein kinase inhibitor (ROCKi), e.g., at a concentration of 5 ⁇ or 10 ⁇ . Exemplary culturing methods for PSCs are reported in Stover and Schwartz, Methods Mol Biol. 2011.
• the PSCs are detached from their maintenance support, e.g., Matrigel, for instance via EDTA treatment.
• EDTA can be used at a concentration of 0.1-10 mM, preferably between 0.5 and 2 mM.
• the EDTA treatment is performed for 1-10 minutes, preferably between 4-6 minutes.
• Preferred EDTA treatment conditions are 0.5 mM EDTA for 5-10 minutes at room temperature and 2 mM EDTA for 5-8 minutes at room temperature.
• step B Culturing the PSCs embedded in a matrix immersed in cell culture medium
• the provided PSCs described above are subsequently cultivated with a matrix, which allows embedding of the PSCs into a 3D environment that is defined by the matrix.
• the embedding and self-organization of PSCs within such a 3D environment provides the basis for the organoid of the invention to be generated.
• the cells should be embedded in the matrix in a homogenous manner.
• the matrix of the invention constitutes a 3D environment that facilitates self- organization and differentiation of the PSCs and cells derived from PSCs.
• the structure of the matrix is stabilized by interactions of the individual matrix molecules, e.g., protein-protein interactions.
• the matrix of the invention forms a hydrogel structure.
• Exemplary suitable matrices of the present invention are collagens, collagen mimics, alginate, fibrin, Matrigel, and chitosan.
• Preferred matrices are collagen and collagen mimics.
• Most preferred matrices are based on collagen type I.
• Matrices based on collagen type I may contain traces of other collagens such as collagen type III.
• matrices based on collagen type I contain more than 80%, more than 85%, more than 90%, more than 95%, or 100% of collagen type I.
• the matrix of the present invention does not comprise Matrigel.
• the present matrix does not comprise Matrigel or other components of natural origin having a non-defined, or ill-defined composition of one or more components. This is particularly important since the use of a non- or ill-defined chemical mixture, such as Matrigel, inevitably results in larger phenotypic variations of the generated organoids. This observation has been confirmed by, for example, Tiburcy et al, Circulation, 2017.
• Matrigel is a non-defined mixture comprising laminin, collagen IV, heparin sulfate proteoglycans, entactin, and growth factor components that are derived from Engelbreth- Holm-Swarm mouse sarcomas, wherein the lot-to-lot component percentages exhibit high variability (Kleinman et al., Biochemistry, 1982).
• PSCs and matrix elements are mixed in a cell culture medium appropriate for PSC culture. Examples for such cell culture media are TeSR-E8 Basal medium (Stemcell) and StemFlex Medium (Gibco).
• the cell culture medium can be supplemented with additional components such as FGF-2 and ROCKi. These components may enhance cell survival and proliferation in the matrix.
• TeSR-E8 Basal medium supplemented with 20 ng/ml FGF-2 and 10 ⁇ ROCKi.
• Other components that may be added to the cell culture medium include DMEM cell culture medium or NaOH for pH neutralization.
• the matrix (such as a collagen matrix) is employed at an effective concentration.
• the concentration of the matrix is between 0.05 mg/ ml and 50 mg/ ml. In other preferred embodiments, the concentration of the matrix is between 0.1 mg/ml and 10 mg/ml. In more preferred embodiments, the concentration of the matrix is between 0.5 mg/ml and 5 mg/ ml. In a most preferred embodiment, the concentration of the matrix is 1 mg/ ml.
• the density of the PSCs following mixture with the medium and matrix can be in the range of 0.1-lOxlO 6 cells per ml.
• a preferred range is 0.5-6 xlO 6 cells per ml.
• a more preferred range is 1-4 xlO 6 cells per ml.
• One exemplary suitable value is 3x10 6 cells per ml.
• step B The present culturing step is performed prior to the culturing step disclosed in the following section.
• This culturing step (where the PSCs are embedded in a matrix; "step B") is performed before a culturing step in which the cells are cultured in cell culture medium comprising a Rho-associated kinase inhibitor (ROCKi) and FGF-2 ("step C").
• ROCKi Rho-associated kinase inhibitor
• step C The transition of step B to step C is typically characterized by addition of the components of step C to the existing cell culture medium.
• this culturing step B is performed for a time period of between 1 minute and 1 day. In preferred embodiments, step B is performed between 5 minutes and 5 hours. In more preferred embodiments, step B is performed between 10 minutes and 1 hour. In even more preferred embodiments, step B is performed between 15 minutes and 30 minutes. In a most preferred embodiment, step B is performed for 20 minutes.
• step B The time point of step B is termed "day -1" by the instant protocol relating to the production of the inventive organoids.
• Step B is performed in a suitable cell culture vessel.
• An exemplary suitable cell culture vessel is a 96-well plate with U-bottom and low attachment properties.
• Step B is performed under conditions that are suitable for PSC survival.
• exemplary suitable conditions are 37°C, 5% C0 2 , for instance, in a cell culture incubator.
• step C Culturing the PSCs in said matrix of step (B) in cell culture medium comprising a Rho- associated kinase inhibitor (ROCKi) and FGF-2 ("step C")
• the PSCs in the matrix of step B are cultivated in medium comprising a Rho-associated kinase inhibitor (ROCKi) and Fibroblast Growth Factor-2 (FGF-2).
• Suitable ROCKi variants include Y27632 (Stemgent), Fasudil, Ripasudil, RKI-1447, GSK429286A, Y-30141, in addition to other components reviewed in Feng et al., J Med Chem., 2016).
• the ROCKi is employed at an effective concentration.
• a preferred ROCKi is Y27632.
• the concentration of Y27632 is between 0.1 ⁇ and 1 mM.
• the concentration of Y27632 is between 1 ⁇ and 100 ⁇ .
• the concentration of Y27632 is between 5 ⁇ and 50 ⁇ .
• the concentration of Y27632 is 10 ⁇ .
• FGF-2 (also known as bFGF) is employed at an effective concentration.
• the concentration of FGF-2 is between 0.1 ng/ml and 1 ⁇ g/ml.
• the concentration of FGF-2 is between 1 ng/ml and 100 ng/ml.
• the concentration of FGF-2 is between 5 ng/ml and 50 ng/ml.
• the concentration of FGF-2 is 10 ng/ml.
• the invention can also be carried out using a FGF-2 mimetic having an equal or similar signaling activity as FGF-2, which is characterized by binding to FGF- receptors to thereby cause FGF-receptor-mediated signaling, wherein such activity is at least 10% of the signaling activity of FGF-2 at each FGF-receptor.
• the PSCs are cultured in a cell culture medium that is appropriate for culturing of the PSCs.
• suitable cell culture medium include TeSR-E8 Basal medium (Stemcell) and StemFlex Medium (Gibco).
• step B This culturing step is performed following step B disclosed above.
• the transition of step B to step C is typically characterized by the addition of the components of step C to the existing cell culture medium comprising PSCs and matrix of step B.
• Step C is performed prior to step D, which is described below.
• the transition of step C to step D is characterized by a partial or complete exchange of the cell culture medium or, alternatively, addition of the components of step D to the existing cell culture medium.
• step C is performed for a time period of between 6 hours and 4 days. In some preferred embodiments, step C is performed between 12 hours and 3 days. In more preferred embodiments, step C is performed between 1 day and 2 days. In a most preferred embodiment, step C is performed for 1 day.
• Step C should begin on day -1 of the protocol for producing organoids. Depending on its duration, this step may extend to day 0, 1, 2, or 3 of the instant protocol. In a most preferred embodiment, step C extends to day 0 of the protocol.
• Step C is performed in a suitable cell culture vessel.
• An exemplary suitable cell culture vessel is a 96-well plate with U-bottom and low attachment properties. Generally, the cell culture vessel will not change when performing steps B and C disclosed herein.
• Step C is performed under conditions suitable for PSC survival.
• exemplary suitable conditions include 37°C, 5% C0 2 , for instance, in a cell culture incubator.
• step D Culturing forming BENQs originating from PSCs and matrix of step (C) in a cell culture medium comprising retinoic acid and one or more inhibitors of SMAD signaling to induce neurogenesis
• the PSCs treated according to step C in the matrix constitute a forming BENO.
• This forming BENO originating from the PSCs and matrix of step C is cultivated in a medium comprising retinoic acid and one or more inhibitors of SMAD signaling pathways. Such treatment induces neurogenesis.
• Retinoic acid is employed as a signaling- activating molecule.
• all-trans retinoic acid [(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-l-yl)nona-2,4,6,8-tetraenoic acid] is used.
• the invention can also be carried out using retinoic acid derivatives having an equal or similar signaling activity as all-trans retinoic acid, which is typically characterized by binding to the retinoic acid receptor to thereby cause retinoic-acid-receptor-mediated signaling, wherein such activity is at least 10% of the signaling activity of all-trans retinoic acid.
• Retinoic acid is employed at an effective concentration.
• the concentration of retinoic acid is between 0.01 ⁇ and 100 ⁇ .
• the concentration of retinoic acid is between 0.1 ⁇ and 10 ⁇ .
• the concentration of retinoic acid is between 0.5 ⁇ and 5 ⁇ .
• the concentration of retinoic acid is 1 ⁇ .
• SMAD inhibitors are employed to inhibit signaling mediated by Mothers against decapentaplegic homologue (SMAD) proteins (e.g., SMAD-1 - SMAD-9). Inhibition of SMAD- mediated signaling induces neurogenesis.
• SMAD inhibitors include the protein noggin, SB 431542 (Tocris), dorsomorphin, and LDN-193189 (Tocris). SMAD inhibitors are employed at an effective concentration.
• noggin can be employed at a concentration of 0.1 ng/ml - 1 ⁇ g/ml, preferably 1 ng/ml - 500 ng/ml, more preferably 10 ng/ml - 200 ng/ml, most preferably 50 ng/ml.
• SB 431542 can be used at a concentration of 0.1 ⁇ - 1 mM, preferably 1 ⁇ - 100 ⁇ , more preferably between 5 ⁇ and 50 ⁇ , most preferably 10 ⁇ .
• the use of more than one SMAD inhibitor may have positive effects on inducing neurogenesis (Example 1).
• One preferred combination of SMAD inhibitors comprises noggin and SB 431542.
• a more preferred combination of SMAD inhibitors consists of noggin and SB 431542.
• a most preferred combination of SMAD inhibitors consists of noggin (50 ng/ml) and SB 431542 (10 ⁇ ).
• the forming BENOs are cultured in any cell culture medium appropriate for PSC culturing.
• Exemplary cell culture media include Stemdiff neuron differentiation medium (Stemcell), Neurobasal Medium (Gibco), and the medium used in Example 2.
• Step D of the disclosed methods is typically performed after step C.
• the transition of step C to step D is characterized by a partial or complete exchange of the cell culture medium or, alternatively, addition of the components of step D to the existing cell culture medium.
• Step D is typically performed before step E, as described herein.
• the transition of step D to step E is characterized by a partial or complete exchange of the cell culture medium or, alternatively, addition of the step E components to the existing cell culture medium.
• step D is performed for a time period of between 2 days and 16 days. In some preferred embodiments, step D is performed between 4 days and 12 days. In more preferred embodiments, step D is performed between 6 days and 10 days. In a most preferred embodiment, step D is performed for 8 days. It was found that performing step D for 8 days is favorable compared to 3 or 6 days (see example 8). Performing step D for 10 days is even more favorable than 8 days, while further extension of step D did not significantly improve the results (see example 8). Therefore, in another particularly preferred embodiment, step D is performed for at least 8 days or for 10 days.
• step D The day “designations" in performing this step depend on the duration of previously performed steps B, C and D.
• the starting point of step D is between days 0 and 3.
• the end of step D is generally between day 2 and day 19.
• step D extends from day 0 to day 8.
• step D extends from day 0 to day 10.
• Step D is performed using any suitable cell culture vessel.
• suitable cell culture vessels include a 96-well plate with a U-bottom and low attachment properties or a 6-well plate or a custom-made 3D-printed or cast molded vessel for single or multi organoid culture.
• the cell culture vessel will typically not change between steps C and D.
• the cell culture vessel may be changed when performing step D.
• One such exemplary vessel change is the expansion from a 96-well plate to a 6-well plate.
• Step D is performed under conditions amenable to forming BENO survival.
• exemplary suitable conditions are 37°C, 5% C0 2 , for instance, in a cell culture incubator.
• step E Culturing the forming BENO of step D in cell culture medium comprising TGF-beta and FGF-2 to enhance the genesis of stromal cells and neurogenesis
• the forming BENO provided following step D is cultivated in medium comprising transforming growth factor (TGF) beta and fibroblast growth factor-2 (FGF-2).
• TGF transforming growth factor
• FGF-2 fibroblast growth factor-2
• Treatment with TGF-beta enhances the genesis and functionality of stromal cells in the emerging organoid.
• the stromal cells are stromal cells of neural tissue. Exemplary stromal cells include glial cells.
• TGF-beta employed in the present invention can be TGF-beta 1, TGF-beta 2, TGF-beta 3, or mixtures thereof.
• TGF-beta mimetic having an equal or similar signaling activity as TGF-beta, which is characterized by binding to a TGF-beta receptor to thereby cause TGF-beta-recep tor-mediated signaling, wherein such activity is at least 10% of the signaling activity of TGF-beta 1.
• TGF-beta employed in the present invention is TGF-beta 1.
• TGF-beta is employed at an effective concentration.
• the concentration of TGF-beta is between 0.1 ng/ml and 100 ng/ml.
• the concentration of TGF-beta is between 0.3 ng/ml and 30 ng/ ml.
• the concentration of TGF-beta is between 1 ng/ ml and 10 ng/ml.
• the concentration of TGF-beta is 5 ng/ml.
• FGF-2 (also known as bFGF) is employed at an effective concentration.
• the concentration of FGF-2 is between 0.1 ng/ml and 1 ⁇ g/ml.
• the concentration of FGF-2 is between 1 ng/ml and 100 ng/ml.
• the concentration of FGF-2 is between 5 ng/ml and 50 ng/ml.
• the concentration of FGF-2 is 10 ng/ml.
• the invention can also be carried out using a FGF-2 mimetic having an equal or similar signaling activity as FGF-2, which is characterized by binding to FGF- receptors to thereby cause FGF-receptor-mediated signaling, wherein such activity is at least 10% of the signaling activity of FGF-2 at each FGF-receptor.
• the forming BENO is cultured in a cell culture medium appropriate for culturing PSCs.
• exemplary cell culture media include Stemdiff neuron differentiation medium (Stemcell), Neurobasal Medium (Gibco), and the medium used in Example 2.
• Step E is typically performed following step D.
• the transition of step D to step E is characterized by a partial or complete exchange of the cell culture medium or, alternatively, addition of the components of step E to the existing cell culture medium.
• Step E is typically performed before step F, which is described below.
• the transition of step E to step F is characterized by a partial or complete exchange of the cell culture medium or, alternatively, addition of the components of step F to the existing cell culture medium.
• step E is performed for a time period of between 2 days and 16 days. In preferred embodiments, step E is carried out between 4 days and 12 days. In more preferred embodiments, step E is performed between 6 days and 10 days. In a most preferred embodiment, step E is performed for 7 days. It was found that performing step E for 5 days is not significantly inferior compared to 7 days; conversely, performing step E for 2 days resulted in inferior BENO condensation (see example 8). Therefore, in another particularly preferred embodiment, step E is performed for at most 7 days or for 5 days.
• step E The day “designations" in performing this step depend on the duration of previously performed steps B, C, D and E.
• the starting point of step E is between days 3 and 15.
• the end of step E is between day 5 and day 20.
• step E extends from day 8 to day 15.
• step E is performed from day 10 to day 15.
• Step E is performed using any suitable cell culture vessel.
• suitable cell culture vessels include a 6-well or a custom-made 3D-printed or cast molded vessel for single or multi organoid culture.
• the cell culture vessel will typically not change between steps D and E.
• the cell culture vessel may be changed when performing step E.
• Step E is performed under conditions amenable to forming BENO survival.
• exemplary suitable conditions are 37°C, 5% C0 2 , for instance, in a cell culture incubator.
• step F Culturing the forming BENO of step E in cell culture medium comprising TGF-beta and one or more inhibitors of notch signaling to enhance genesis of stromal cells and neurodifferentiation.
• the forming BENO provided following step E is cultivated in medium comprising transforming growth factor beta (TGF-beta) and one or more inhibitors of notch signaling.
• TGF-beta transforming growth factor beta
• Treatment with TGF-beta enhances the genesis of stromal cells, while inhibition of notch signaling enhances neurodifferentiation.
• TGF-beta employed in the present invention can be TGF-beta 1, TGF-beta 2, TGF-beta 3, or mixtures thereof.
• the invention can also be carried out using a TGF-beta mimetic having an equal or similar signaling activity as TGF-beta, which is characterized by binding to a TGF-beta receptor to thereby cause TGF-beta-recep tor-mediated signaling, wherein such activity is at least 10% of the signaling activity of TGF-beta 1.
• TGF-beta is employed at an effective concentration.
• the concentration of TGF-beta is between 0.1 ng/ml and 100 ng/ml. In preferred embodiments, the concentration of TGF-beta is between 0.3 ng/ml and 30 ng/ml. In more preferred embodiments, the concentration of TGF-beta is between 1 ng/ ml and 10 ng/ ml. In a most preferred embodiment, the concentration of TGF- beta is 5 ng/ ml.
• Notch signaling inhibitors are employed for inhibiting cell signaling mediated by a notch receptor (e.g., notch 1 - notch 4 receptors). Inhibition of notch signaling enhances neurodifferentiation.
• Suitable notch signaling inhibitors include JV-[(3,5-Difluorophenyl)acetyl]-L- alanyl-2-phenyl]glycine-l,l-dimethylethyl ester (DAPT), Compound E (Stem cell technologies), and ⁇ -secretase inhibitors such as those described in Olsauskas-Kuprys et al., OncoTargets and Therapy, 2013. Notch signaling inhibitors are employed at an effective concentration.
• a preferred notch signaling inhibitor of the present invention is DAPT.
• the concentration of DAPT is between 0.01 ⁇ and 100 ⁇ . In some preferred embodiments, the concentration of DAPT is between 0.1 ⁇ and 10 ⁇ . In more preferred embodiments, the concentration of DAPT is between 0.5 ⁇ and 5 ⁇ . In a most preferred embodiment, the concentration of DAPT is 2.5 ⁇ .
• the forming BENO is cultured in a cell culture medium that is appropriate for its culturing.
• exemplary cell culture media include Stemdiff neuron differentiation medium (Stemcell), Neurobasal Medium (Gibco), and the medium used in Example 2.
• Step F is typically carried out following step E.
• the transition of step E to step F is characterized by a partial or complete exchange of the cell culture medium or, alternatively, addition of the components of step F to the existing cell culture medium.
• step F is performed for a time period of between 5 days and 95 days. In preferred embodiments, step F is performed between 7 days and 50 days. In more preferred embodiments, step F is performed between 10 days and 20 days. In a most preferred embodiment, step F is performed for 13 days.
• step F The day “designations" in performing this step depend on the duration of previously performed steps B, C, D, E and F. In general, the starting point of step F is between days 4 and 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.
• One exemplary suitable cell culture vessel is a 6-well plate or a custom-made 3D-printed or cast molded vessel for single or multi organoid culture.
• the cell culture vessel will typically not change between steps E and F.
• Step F is performed under conditions suitable for forming BENO survival.
• exemplary suitable conditions are 37 °C, 5% C0 2 for instance, in a cell culture incubator.
• step F of the methods disclosed herein provides a BENO.
• the BENO may be further cultured or expanded under suitable conditions, depending on the desired application of the BENO. Additional culturing may lead to the development of additional features of the BENO and/ or optimize neurogenesis.
• Neuronal organoids produced by the methods disclosed herein present cortical development, neurogenesis and gliogenesis.
• the BENOs comprise neuronal cells and stromal cells.
• Stromal cells are important for providing a neurogenesis-promoting environment that is created upon practicing the disclosed methods. Concurrent neurogenesis and gliogenesis via the defined growth factors and small molecules disclosed herein effectively reconstruct the multicellular complexity of the human brain.
• Neuronal organoids produced by the disclosed methods reveal unique features that have not been previously shown.
• the BENOs described herein form functional neuronal networks characterized by neuronal functionality, including the formation of functional synapses, the formation of hierarchical networks, GABAergic networks, and also the synchronization of neurons (Example 5). These neuronal capabilities represent at least one important advantage over conventional neuronal organoids.
• BENOs can comprise excitatory and inhibitory neurons and excitatory and inhibitory neuronal networks.
• BENOs produced by methods of the present invention are preferably produced under fully defined conditions (e.g., serum-free). This means that the BENOs are reliably reproducible since variations originating from non- or ill-defined chemical components is eliminated.
• BENOs produced by methods of the present invention can be used for so-called phenotypic drug screening.
• phenotypic drug screening is not focused on the binding of a candidate molecule to a specific target, but instead on the effect that a target molecule has on a phenotype.
• a pre-condition of such phenotypic drug screening is the presence of an appropriate model that can mimic a phenotype of the investigated disease.
• the presently disclosed BENOs may provide such a disease model when investigating various diseases related to neural tissues.
• Diseases for which the present invention can provide a suitable model for drug screening include stroke, brain inflammation disorders, neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease, per Example 6), neuroinflammatory diseases (e.g., multiple sclerosis), traumatic injury (e.g., brain- surgery-induced injury), channelopathy (e.g., epilepsy) and psychiatric diseases (including autism and schizophrenia, per Example 7).
• neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, per Example 6
• neuroinflammatory diseases e.g., multiple sclerosis
• traumatic injury e.g., brain- surgery-induced injury
• channelopathy e.g., epilepsy
• psychiatric diseases including autism and schizophrenia, per Example 7).
• BENOs can be used for discovery and drug refinement by phenotypic drug screening. This use of BENOs includes the discovery and refinement of drugs that may induce or enhance repair, regeneration, protection, and disease prevention in brain and neural tissue.
• the PSCs useful for such BENO models can be obtained from healthy individuals or from diseased patients.
• gene editing of pluripotent stem cells can be applied to create any genetic and epigenetic modification of interest.
• the BENOs After producing BENO compositions from the PSCs according to the presently disclosed methods, the BENOs thus allow phenotypic tissue screening with high predictive value. Due to the higher degree of maturation, cellular complexity, and hierarchical network function of neural tissues, using the BENOs according to the invention offers numerous advantages over conventional models. Moreover, the simplicity of the disclosed methods for BENO production readily enables high-throughput phenotypic screening. Healthy and disease-modelled BENOs can be subjected to simulated diseases (for example, hypoxia to induce stroke-like damage).
• Phenotypic read-outs include effects on tissue formation, electrical connectivity, cell death and cell proliferation in specific cell types within BENOs.
• a phenotypic drug screen can allow the definition and validation of a variety of drug targets. This can thus provide a basis for subsequent compound screening for identifying, e.g., compounds having regenerative, reparative, disease modifying, or protective biological activity.
• BENOs produced according to the presently disclosed methods can also be used for drug safety screening to test, for instance, the potential of a substance to induce electrical disturbances (seizures), degeneration, cell death, or other cellular anomalies in neural tissue.
• BENOs produced by methods of the present invention can also be used for mode of action studies involving drugs, e.g., in preclinical trials running in parallel to clinical trials.
• BENOs produced by methods of the present invention can also be used for personalised medicine purposes.
• patient-derived iPSCs can be used to simulate diseases and test personalized therapies.
• BENOs of the present invention could be particularly useful in testing therapies for diseases that are related to neural tissues, e.g., neurodegenerative diseases or neuroinflammatory diseases. All possible therapy options could be explored and tested using the described BENOs, e.g., therapies by drugs, biological agents like antibodies or non-coding RNAs, gene editing, or a combination thereof.
• BENOs can also be applied to generate increasingly elaborate tissue models in co- cultures of BENOs with other tissue engineering platforms.
• co-culture with EHM Engine Heart Muscle
• BSMs biologically modified skeletal muscle
• co-cultures of BENOs with EHM can be used to study neuron and pacemaker or neuron and cardiomyocyte interactions. This model can then be used to study arrhythmia development.
• BENOs are co- cultured with ESM (Engineered Skeletal Muscle), ELT (Engineered Liver Tissue), or ECT (Engineered Connective Tissue).
• BENO-tumor models are used to study tumor-brain interactions (e.g., tumor brain invasion, metastases spread) or BENO -leukocyte infiltration models are used to study neuronal inflammation (e.g., autoimmune diseases).
• tumor-brain interactions e.g., tumor brain invasion, metastases spread
• BENO -leukocyte infiltration models are used to study neuronal inflammation (e.g., autoimmune diseases).
• co-culture models represent an important step towards parallel multiple organ screening for drug action and interaction.
• Co-culture models can be further used as models for diseases such as Parkinson's and other neurodegenerative diseases, e.g., to test their efficacy as a personalized treatment.
• the BENOs of the present invention may also provide regenerative tissue for scientific or therapeutic purposes.
• BENOs may be injured to study recovery and regeneration under drug or biophysical (e.g., electrical conditioning) treatment.
• BENOs may be constructed with specific brain functions, such as dopamine production and release to counter Parkinson's disease.
• Further BENOs may be connected to organs or used as machine-organ interfaces to enable control of enervated organs (e.g., control of skeletal muscle).
• Kits of the present invention include components for practicing the presently disclosed methods.
• a kit contains PSCs, a matrix, suitable media and the required supplements (ROCKi, FGF-2, retinoic acid, one or more inhibitors of SMAD signaling, TGF- beta, and one or more inhibitors of notch signaling).
• a kit contains a matrix, suitable media and the required supplements (ROCKi, FGF-2, retinoic acid, one or more inhibitors of SMAD signaling, TGF-beta, and one or more inhibitors of notch signaling).
• a kit contains a matrix and the required supplements (ROCKi, FGF-2, retinoic acid, one or more inhibitors of SMAD signaling, TGF-beta, and one or more inhibitors of notch signaling). In other embodiments, a kit contains a matrix and at least 4 of the required supplements (ROCKi, FGF-2, retinoic acid, one or more inhibitors of SMAD signaling, TGF- beta, and one or more inhibitors of notch signaling).
• FGF-2 stock at 10 ⁇ g/ml prepared according to manufacturer's instructions
• Noggin stock at 250 ⁇ g/ ml prepared according to manufacturer's instructions
• TGFB1 stock at 10 ⁇ g/ml prepared according to manufacturer's instructions
• Matrigel 1:30 Solution Dilute 1 ml Matrigel in 29 ml ice cold PBS using pre-cooled tips in order to protect Matrigel from polymerizing. If solution is not clear store in the fridge overnight and mix again.
• Basal medium 50 ml supplemented with:
• NPEM Neural progenitor expansion medium
• NDM Neural Differentiation Medium
• Day -4 standard maintenance culture of pluripotent stem cells, employed as an example for the preparation of cells
• Growth factor reduced Matrigel 1:120 was diluted in ice cold PBS and plates were immediately coated (150 ⁇ /crrr). The coated plates were then stored in a refrigerator for a maximum of 2 weeks. Prior to use, the coated plates were disinfected with ethanol and placed at 37°C in an incubator for 30-60 minutes.
• FGF-2 treatment at day -2-0 did not appear to provide any advantage compared to untreated tissues.
• treatment of the cells with FGF-2 at days 8-15 i.e. NPEM use
• markedly induced proliferation of the cells including NPCs which resulted in more complex neuronal networks at day 28, as visualized by IF whole mount staining of neurofilament (FIG. 3B).
• Double treatment (day -2-0 and days 8-15) did not seem to be advantageous over a single treatment (days 8-15).
• protocol 4 the proliferative effects of TGF-beta 1 for glia cells (day 8-28) were tested.
• protocol 5 TGF-beta 1 and DAPT treatments were combined in order to test whether development of glia and neurons can be enhanced simultaneously.
• the different protocols are summarized in FIG. 6A.
• protocol 3 protocol 3
• protocol 5 protocol 5
• DAPT and TGF-beta protocol 3
• the expression of neuronal markers decreased with the time in both protocols, suggesting that other cell types may arise, an expected result since from day 28 the tissues are no longer undergoing treatment.
• protocol 5 clearly supported gliogenesis as evidenced by a 10-fold increase of GFAP transcripts.
• Protocol 5-derived tissues contained more glia than those derived from protocol 3, whereas the amount of mature neurons appeared to be the same.
• the collective data support a conclusion that, upon dual SMAD inhibition in the presence of RA, FGF-2 treatment enhances NPCs, and DAPT supports neurogenesis while TGF-beta support gliogenesis. Accordingly, the optimized differentiation protocol (Example 2) incorporates treatment using all of the above-described factors (time points indicated in FIG. 6A).
• EXAMPLE 2 Optimized protocol for generating human bioengineered neuronal organoids (BENOs)
• BENOs human bioengineered neuronal organoids
• NCM Neural Commitment Medium
• Basal medium 50 ml supplemented with 10 ⁇ SB 431542 (50 ⁇ ), 50 ng/ml noggin (10 nl), and 1 ⁇ RA (5 ⁇ ).
• Neural progenitor expansion medium (NPEM) [00222] Basal medium (50ml) supplemented with 10 ng/ml FGF-2 (50 ⁇ ) and 5 ng/ml TGFB1.
• NDM Neural Differentiation Medium
• Basal medium 50 ml supplemented with 2.5 ⁇ DAPT (1.25 ⁇ ) and 5 ng/ml TGFB1 (25 ⁇ ).
• Day -4 standard maintenance culture of pluripotent stem cells, employed as an example for the preparation of cells
• neuronal receptor expression GABA, DOPA, Kainate, AMP A, NMD A
• ion channel expression K + , Ca 2+ , Na +
• synaptic protein expression SYN1, SYT4, SYP, SYNPO
• CTIP2 Layer V-VI neurons
• TBR2 subventricular zone precursor cells
• SOX2 neural stem cells
• GABAergic interneurons were validated. Staining for both the GABAergic marker GABA and the respective receptor GABBR2 (FIG. 9A, FIG. 9B) was performed. GABA was strongly expressed in the soma compartments as well as at the synaptic boutons in the axons of GABAergic neurons. GABBR2 was observed to be expressed in neuronal perikarya. Moreover, an elaborate dopaminergic network was immunostained with TH, thereby indicating a midbrain identity.
• EXAMPLE 5 Neuronal activity: GABAergic network
• Examples 1-5 of the present invention convincingly demonstrate novel collagen-based, serum-free, stage-specific directed differentiation methods for the engineering of electrically active neuronal networks from human pluripotent stem cells.
• EXAMPLE 6 BENOs as a model for neuronal regeneration
• Cryo-, mechanical-, hypoxia-, or neurotoxin-induced global or local injury can be produced for example by a N 2 -cooled metal pin, compression or dissection, culture in subcritical to 0% oxygen, as well as exposure to glutamate, dopamine, ethanol, tetrodotoxin, botulinum or tetanus toxins.
• Recovery from injury including cell specific responses e.g., by neurons and glia cells
• Repair and regeneration for example by induction of neuronal proliferation and protection from injury for example by application of antioxidants or pharmacological modulators are studied.
• BENOs serve as models to identify and validate novel targets for disease modification including neuronal protection, repair, and regeneration.
• BENOs are used at different stages of development, e.g., as proliferating neurons (day 15, day 28 of the Example 2 protocol) and post-mitotic neurons (day 40, day 60 of the Example 2 protocol).
• EXAMPLE 7 BENOs as personalized disease model, e.g., for schizophrenia
• BENOs are created from pluripotent stem cells, for example derived from patients by reprogramming or genetically engineered for example by CRISPR/ Cas or TALEN technologies.
• BENOs are phenotyped as described above using means of calcium activity assessment, immunofluorescence analysis, or electrophysiological means (e.g., impaling electrode measurements, multi-electrode arrays/field potential measurements).
• electrophysiological means e.g., impaling electrode measurements, multi-electrode arrays/field potential measurements.
• RNA sequencing and proteome analyses is conducted for phenotype-genotype association studies.
• BENOs A use of BENOs is modeling aspects of schizophrenia.
• BENOs are created from iPSC cells obtained from schizophrenic patients. Subsequently, BENOs from these patients are phenotyped to analyze the phenotypic differences to healthy control subjects. Other studies are designed to investigate BENOs derived from patients suffering from autism, familiar hemiplegia and epilepsy.
• EXAMPLE 8 Further optimization of neuronal induction and expansion in steps C and D
• DAPT notch response inhibitor

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