FUNCTION AND DISORDERS

Field of invention

This disclosure relates to tissue engineering, more particularly cell culture devices, i.e., organs-on-chips, organoids and neurospheres.

BACKGROUND

Techniques for creating organs-on-chips are known. Thus, a person of ordinary skill in the art understands how to create three-dimensional cell cultures on a small“chip” to simulate biological characteristics of living organs. The techniques employ those that have been developed for lab-on-chip and cell cultures as well as tissue engineering.

Specifically, brain organoids have been created by culturing stem cells in a bioreactor to study disease and brain function and development. For example, brain organoids were described by Lancaster and Knoblich in 2014 (Science. 2014 Jul 18; 345(6194):1247125. doi: 10. H26/science.1247125. Epub 2014 Jul 17.). Aggregates of neurons have been observed, for example, See Segev et al Phys Rev Lett. 2003;90(16):168101. doi:

l0.l l03/PhysRevLett.90.l68l0l. PubMed PMID: 12732015.

The current state-of-the-art brain microphysiological system (MPS) approaches (“brain- in-a-dish”), including brain organoids, are still not capable of capturing the inherent functional complexities of brain, and its complex disorders. To address these short comings, the disclosed subject matter provides a novel hierarchic neural MPS approach, i.e., a network of neurospheres (NNet).

SUMMARY

This disclosure relates to a neurosphere network that contains a plurality of artificial neurospheres on a surface, wherein the neurospheres are interconnected by axons and optionally comprise a mean for measuring neuronal activity and developmental processes. The neurosphere network may be 2D or 3D. This disclosure also relates to a method of making a neurosphere network by applying neuronal cells to a non-adhesive surface of polysaccharide, such as agarose, or of a silicone-based organic polymer, wherein the neuronal cells on the non-adhesive surface are in cell groups spaced apart from each other, and growing the neuronal cells under culture conditions to form the cell groups into neurospheres, and such that axons form inter-connecting the spaced-apart cell groups or neurospheres, to form a neurosphere network. The cell groups are thus self- aggregated assemblies.

In an embodiment, the neuronal cells are dissociated from mammalian embryonic hippocampus/cortex, or are human iPSC-derived neuroprogenitors cells (NPCs).

The neurospheres may be derived from neuronal cells that are of the same cell type or that are of different cell types. The resulting neurosphere network may contain interconnected neurospheres that represent different tissue types, e.g., corresponding to different parts of the brain.

Optionally, the individual neurospheres are collected and re-positioned strategically on the adhesive surface. The inter-connections may form passively or actively by using guidance cues like Netrins (e.g., using soaked microbeads).

Neurospheres may be spaced apart from each other by substantially the same distance or by varying distances. The distance(s) may vary from mhi to mm. In embodiments, the distances may be 1 mm, in further embodiments, the distance may be at least 2 mm. In embodiments, the distances may be at least 5 mm. In embodiments, the distances may be at least 10 mm. In embodiments, the distances may be at least 15 mm. In embodiments, the distances may be a maximum of 1 mm, in further embodiments, the distance may be a maximum of 2 mm. In embodiments, the distances may be a maximum of 5 mm. In embodiments, the distances may be a maximum of 10 mm. In embodiments, the distances may be a maximum of 15 mm. In an embodiment, the distance(s) are determined by self organization, and in other embodiment by the strategic positioning of the individual neurospheres on the adhesive surface. The neuronal cells may contain a means for measuring neuronal activity, such as a viral vector, a calcium sensitive dye or protein, or an array of electrodes.

In an embodiment, the non-adhesive surface is a plurality of microwells or is a flat mold. The non-adhesive surface may be a polysaccharide, such as agarose. Alternatively, the non-adhesive surface may be a silicone-based organic polymer, such as

polydimethylsiloxane (PDMS). Preferably, the non-adhesive surface is in the form of agarose-based microwells or a flat PDMS mold that are casted in 3D printed stencil mold.

In embodiments, the neurospheres are collected after at least 10 hours after plating, or optionally 12, 14, 16, 24 or more hours after plating to establish the axon inter connections between neurospheres. In self-organized embodiments, neurospheres axon inter-connections are spontaneously formed.

In embodiments, NNet mimics the small-world hierarchic-modular architecture of mammalian brains (i.e. highly intra-connected modules with fewer inter-modular connections). NNet are created by synthetically building a network of inter-connected individual brain MPSs.

A method includes growing cells (either dissociated from wild-type or diseased mammalian embryonic hippocampus/cortex, or human iPSC- derived neuroprogenitors cells (NPCs)) on a non-adhesive surface made of polydimethylsiloxane (PDMS), agarose or similar materials (see FIGs. 1 and 2) that facilitates the adhesion of cells to each other rather than to the surface. As a result, the cells self-aggregate into 3D assemblies

(Neurospheres) formed by all neuronal cell-types, which are then inter-connected either passively or actively by axons forming networks of Neurospheres.

In embodiments, the NNet is used to screen drugs by applying a drug of interest to the NNet and measuring the response as neuronal activity code readout over a period of time. In embodiments, the NNet is used to generate quantitative data indicating fundamental brain computation for example by showing magnitude of brain activity by region or under the effect of selected drugs. In embodiments, the NNet is used to observe and quantify the sizes, positions, and activity level of neuronal ensembles against optical or pharmaceutical perturbations. For personalized medicine, a NNet may be grown from iPS cells derived from a specific patient or patient sub-population, and the resulting NNet tested for drug response.

In an embodiment, this disclosure is a method for screening a compound for activity against a brain disorder, by (a) contacting the compound with a first neural network according to claim 1 or obtained by a method according to any one of claims 2 to 22, wherein the first neural network originated from brain cells obtained from a first mammal or derived from stem cells or iPSC cells of a first mammal presenting a disorder or a biomarker indicative of the brain disorder, (b) contacting the compound with a second neural network according to claim 1 or obtained by a method according to any one of claims 2 to 22, wherein the second neural network originated from brain cells, or brain cells derived from stem cells or iPSC cells, of a second mammal of the same species as the first mammal but lacking the brain disorder or biomarker; and (c) recording activity patterns, using calcium imaging or electrophysiology, over time from the first and second neural networks after the contacting steps, and analyzing the activity patterns to determine a difference in generated signal characteristic of the brain disorder. This method may also include a subsequent step of (d) fixating the neural networks after live recording, and staining and visualizing the molecular identity of the cells for a biomarker.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a hierarchic-modular neurosphere network (NNet) for recapitulating the brain complexities.

FIG. 2 illustrates correlated neuronal activity (ensembles) in individual neurosphere vs. NNet preparations.

FIG. 3 shows preparation NNet of specific configuration.

FIG. 4 illustrates a chronic ketamine treatment NNet model of schizophrenia

pathophysiology. FIG. 5 shows loss of correlation (neuronal ensembles) in chronic ketamine treatment NNet model of schizophrenia pathophysiology.

FIG. 6 illustrates modeling epileptic seizures with NNet.

FIG. 7 shows an example of live calcium imaging data that is collected, analyzed to extract position and activities of individual brain cells.

FIG. 8 shows activity traces of cells identified in FIG. 7.

FIG. 9 plots the synchronization of activities of brain cells (quantified as average pairwise correlation).

FIG. 10 to FIG. 14 show example activity patterns at different days (DIVs).

FIG. 15 shows use of a custom miniaturized microscope, placed directly in the incubator for long-term activity recording, and also real-time data compression and analysis.

FIG. 16 is an example of a fixed sample and its staining to visualize molecular and structural details. These are correlated with the recordings from this sample.

FIG. 17 is a comparison of synchrony of a control and SETD1 mutant (model of Schizophrenia).

FIG. 18 to FIG. 21 show activity pattern images of diseased (in top row in FIG. 18, left column in FIGs. 19 to 21) and control (in bottom row in FIG. 18, right columns in FIGs.

19 to 21).

FIG. 22, FIG. 23, and FIG 24 shows activity patterns in developing brains as reported in literature (cited in FIGs).

FIG. 25 to FIG. 33 show results of evaluating the presence of calcium spikes, SPAs, and GDPs in NNets. DETAILED DESCRIPTION

Those skilled in the art will understand that this invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth in this application. Rather, these embodiments are provided so that this disclosure will fully convey the invention to those skilled in the art. Many modifications and other embodiments of the invention will come to mind in one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing description.

The drug development for psychological and neurological disorders has essentially stalled due to high rate of failures in clinical trial. Our NNet approach, which can be derived from patient specific iPSC cells, provides an effective mean for modeling of brain functional complexity and disorders, while providing systems-level observation and manipulation capabilities. NNet, in combination with microfluidics and miniaturized imaging approaches, can yield a compact and effective screening platform for psychoactive compounds. In addition, we expect NNet to also inspire next generation of machine learning approaches by discovering principles of higher order brain function.

Compared to brain organoids, the disclosed NNet subject matter may provide the following advantages:

Time of production: To grow mature functionally-active organoids takes around 4-6 months while NNet will take only a matter of days or weeks.

Accessibility: Brain organoids are opaque and dense, making very difficult to study the internal connectome organization. The NNets exposes all the connections for easy observation and manipulation.

Plasticity: Formation of the functional neuronal networks in Brain organoids are not easy to control, whereas NNet are amenable to rational designs by employing different shape and patterns of molds and/or the guided axonal connectivities (e.g., by using Netrin). Heterogenity: So far, only Bagle et al 2017 (Nat Methods. 2017 Jul;l4(7):743-75l. doi: l0.l038/nmeth.4304. Epub 2017 May 10.) have been able to grow hybrids organoids with two different regions, but the NNet disclosed herein permits combine in even more complex and controlled way using neurospheres derived from different brain regions.

Advantages of the NNet approach may include: (1) arbitrary control of the size and patterns of NNet, and therefore their complexity and correspondence to higher order brain functions; (2) precise manipulation (using optical means) and observation (using large field-of-view imaging) access to individual neurons, connections, modules and even the entire network, and (3) multi-scale intra-/inter-/supra-modular activity and connectivity patterns to better model brains; (4) amenability to automated high-throughput screening platforms (e.g., using microfluidics) for psychoactive compounds; and (5) access to patient-specific analysis.

Such features advance the ability to model brain complexities and its disorders. Being open preparation, NNet are easy to integrate in microfluidics based platform for systematic screening of psychoactive compounds for brain diseases. Patient specific NNet can be sourced from iPSC-derived neuronal progenitors to implement personalized medicine assays. We have also discovered precise sample-invariant quantitative descriptors that correspond to higher-order in vivo brain functions including formation and maintenance of neuronal ensembles, memory storage and recall.

Applications include: (1) modeling of complex brain disorders to understand the pathophysiology of neural circuits at systems level, (2) automated and high-throughput drug screening, (3) personalized medicine for psychiatric disorders, and (4) improving machine learning approaches by combining with artificial neural network theories.

In general, pharma industry will be most interested in using these methods for better screening of potential drugs before clinical trials. In addition, companies aiming for personalized treatment may benefit from quick assays to assess the efficacy of various available options, and this will provide models for basic research on the complex physiology of brain architecture. Self-organized NNet

In its simplest form, NNet can be developed by plating of neural cells on PDMS molds of specific pattern and size. The starting cells can express calcium indicator GCaMPs (using standard AAV vectors or transgenically) as proxy for neuronal activity. Calcium sensitive dyes can be used as alternative. The activity can also be recorded by an array of electrodes. We implemented the NNet and found more complex activity patterns (FIG. 1 and FIG. 2). Due to open-access layout, NNet is amenable to cell-type specific activation/deactivation (e.g., using optogenetic, or light-sensitive caged compounds as described in the accompanying IR) for model brain function and disorders (e.g., Autism Spectrum Disorder is caused by imbalance in Excitation/Inhibition).

Supervised NNet assembly

In this approach, one can design any specific NNet configuration. NNet forms in two steps: cells aggregate to form individual 3D neurosphere/unit, followed by their inter connection. This allows us to collect individual neurospheres and re-position them strategically on a patterned PDMS mold for inter-connections passively or actively by using guidance cues like Netrins (e.g. using soaked microbeads) (see FIG. 3). We found that neurospheres collected at least before 12 hours (after plating) establish inter connections.

Heterogeneous NNet

Supervised NNet allows assembling of heterogeneous NNet by combining individual units from different sources (Figure 3 in attachment), such as normal and disease models, or different brain regions. Such preparations allow effective investigation of brain disorder mechanisms. For example, most brain disorders start in small regions, followed by their spread across the entire brain. This phenomenon can be easily capture by generating heterogeneous NNet composed of only a few nodes from diseased sources. Quantitative descriptors of higher order brain functions

One major problem in the MPS field is how to extract meaningful quantitative descriptors that correspond to higher-order brain functions. We have identified several such features, including: (1) Formation of neuronal ensembles (i.e. correlated cells) which are the unit of computation and information storage. (2) Maintenance of ensembles over time as a measure of stability of information encoding and processing. (3) Pattern completion, by activating part of an ensemble to capture model memory recall. (4) Integration of function and the underlying structure to model causal structure-function relationships.

Applications

The NNet approach may be used for modelling of brain and the disease pathophysiology, personalized medicine by patient-specific NNet for screening of psychoactive drugs, and next generation artificial neural networks.

For drug screening, brain cells may be obtained (or derived from stem cells or iPSC cells) of mammals presenting a biomarker indicative of a brain disorder, where the biomarker may be a gene mutation or a biologically active protein, or any other physiological material such as polynucleotides that indicate a disease state. For example, the biomarker may be a neurotransmitter-related protein or molecule (for example parvalbumin, somatostatin, glutamate, GABA, or dopamine) or a transcription factor or an effector gene marker.

The compound being screened may be a small molecule, a polynucleotide, a protein, or a virus particle. For example, the compound may be a DNA based or RNA based active compound, or a biologically active protein such as an antibody.

The brain disorder against which the compound is screened may be, e.g., schizophrenia, epilepsy, autism, Parkinson’ s disease, depression, or a neurodegenerative brain disorder such as Alzheimer’s disease.

The screening method captures signals (e.g., low average pairwise correlation of activities) characteristic of the brain disorder. The signal may be one or more factors, preferably the patterns of activity of individual brain cells and their evolution over time, synchronization or correlation of activity of all brain cells or brain cells belonging to individual units over time, formation and stability of neuronal ensembles, stable temoral activity sequences, or the population level activity evolution over time, as quantified by dimensionality reduction. The dimensionality reduction may be, e.g., principal component analysis, independent component analysis, or tSNE, or the relationship of activity patterns to underlying molecular identity of brain cells.

As explained above, NNet approach is validated for effective modeling of Schizophrenia pathophysiology (see FIGs. 4 and 5) and epilepsy (see FIG. 6).

The imaging data shown in FIG. 7 resulted from Calcium Imaging data Analysis (using CalmAn based-pipeline). FIG. 8 shows activity traces of cells identified in FIG. 7. In FIG. 8 each row represents a brain cell. Shown on the left of FIG. 8 is a heatmap showing level of activity (i.e. delta F over F, which is a standard way to quantify change in signal). Shown on the right of FIG. 8 is discretization of these signals, i.e., discrete spikes, to identify when a brain cell fires (or becomes active).

FIG. 9 plots the synchronization of activities of brain cells (quantified as average pairwise correlation) and compares two situations: orange when the individual units are not connected to each other, and cyan when individual units are connected to each other. The plot on the left (titled Neurons in Neurosphere) plots average correlation among only those neurons that belong to the same unit. The plot on the right (titled All Neurons in NNet) is for all neurons without any restriction. The bottom-right graph plots average number of firing events. In the plots“DIVs” means days in vitro i.e. days after culturing.

It is notable that in left plot the correlation becomes high very quickly and remain high, whereas in right the correlation increases slowly, and then decreases. Orange is meant to show that this effect is not seen for preparations that are not connected. Activity patterns at different days after culturing (DIVs) are shown in FIG. 10 to FIG. 14. It is notable that even though in higher DIVs the correlation is low, as in the beginning DIVs, the activity is much more structured, indicating development of distinct patterns and connections over time, mimicking developing brain circuits. FIG. 15 shows the results obtained when the experiment was done using a custom-made miniaturized microscope placed directly in an incubator. The live recording was conducted for about 5 mins at high speed at defined intervals (e.g. every 2 hours). The plots on the bottom show one example of pairwise average correlation of one network over time. As noted in FIG. 15, the sample was recorded for about 5 minutes, every 2 hours. Different plots are calculations of average correlation by pooling in time points.

FIG. 17 compares synchrony of a control and a SETD1 mutant (a model of

Schizophrenia). The top graph shows that the diseased ones do not develop high synchrony. FIG. 18 to FIG. 21 clearly showing striking differences in structure of activity patterns for diseased (on the left) and control (on the right, after animation). These differences, i.e., in synchrony and also patterns of activity, can be used to assess if treatment by a specific drug candidate can restore them.

FIG. 22, FIG. 23, and FIG. 24 show that the NNet preparations capture the kinds of patterns that are known from real brains in literature (shown). This demonstrates that one can transfer the knowledge gained from these cultures to brain defects.

FIG. 25 to FIG. 33 show results of evaluating calcium spikes, Synchronous Plateau Assemblies (SPAs), and GDPs in NNets. Cells were loaded with fura 2-a.m. and the slice was imaged using multibeam two-photon excitation with a 20x objective. Acquisition rate was 100 ms/frame. Long-lasting calcium transients were visible in several cells. In FIGs. 26, the three images on the right show the first frame of a representative video.

In FIG. 33, the three images on the right show the first frame of a representative video of spontaneous activity in the mouse CA1 hippocampal region in control conditions at the end of the first postnatal week (P6). Synchronous fast calcium transients are clearly visible.The following examples serve to illustrate certain aspects of the disclosure and should not be construed as limiting the claims. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. EXAMPLES

Example 1: Self-organized NNet

FIG. 1A is a schematic summary of methods for growing individual and network of neurospheres. Neuronal tissues are extracted from cortex/hippocampus of late stage embryos (El 8 or early post-natal stages), followed by Trypsin treatment. Alternatively, human iPSC- derived neural progenitor cells can be used. The dissociated cells are infected with AAVl.Syn.GCaMP6f.WPRE.SV40 vector and plated in agarose-based micro- wells or in a flat PDMS mold (to yield NNet). Micrograph of an example NNet is shown. FIG. IB shows single brain neurospheres exhibiting slow oscillatory activities. dF/F traces were extracted by overlaying datasets with a grid of super-pixels (16x16 microns). The traces are plotted separately in the graph, as identified by numbers 1 and 2. FIG. IB shows activity of single and isolated neurospheres, while FIG. 1C shows that a NNet exhibit much more complex activity patterns. A NNet preparation was imaged for three consecutive days for 4 minutes durations (100 sec data shown for clarity). dF/F traces belonging to particular NNet node are plotted separately, as identified by the colored numbers.

Example 2: Self-organized NNet

FIG. 2 illustrates correlated neuronal activity (ensembles) in individual neurosphere vs. NNet preparations. Neurospheres activity profiles (dF/F) are shown from both individual (top) and NNet (bottom). Heat maps shows the pairwise correlations. As evident from the data, NNet activity is coordinated and synchronized, unlike in individual neurospheres.

Example 3: Preparation NNet of specific configuration

FIG. 3 shows how NNet of a defined composition (i.e. of different types, marked by different colors) and connectivity patterns (and hence complexity) can be derived by first growing individual units in agarose or PDMS mold followed by their placement on a patterned mold made of PDMS or similar material. Neurospheres can establish inter connections either passively, or activity when stimulated by axonal guidance directional cues such as Netrins. Netrins or similar axonal guidance molecules can also be used to control the directionality of the connections, when delivered in spatially restricted manner (e.g., using Netrin soaked microbeads).

Example 4: Chronic ketamine treatment NNet model of schizophrenia

pathophysiology.

NNet preparations were treated with ketamine (10 mM final concentration) every 24- hours. Calcium imaging was performed before adding ketamine and 2 days after. dF/F traces for all cells pre-/post- ketamine treatment are shown in FIG. 4. In FIG. 4A the micrograph on the left shows the NNets. Principal component analysis (PCA) was performed to determine clusters of co-active neurons (i.e. ensembles). All the cells belonging to the top three principal components are color-plotted in sample space, and the corresponding principal component traces are shown. As evident, identified ensembles before treatment were affected (marked by yellow arrows). Similarly, in FIG. 4B, dF/F traces and PC components are shown for a time control series. As evident, the spatial structure of ensembles remains intact, even though the temporal profiles have changed, similar to observations in mice. These experiments provide strong support for the feasibility of using NNet for modelling of schizophrenia pathophysiology.

Example 5: Loss of correlation (neuronal ensembles) in chronic ketamine treatment NNet model of schizophrenia pathophysiology.

NNet preparations were treated with ketamine (10 mM final concentration) or saline control every 24-hours. Calcium imaging was performed before adding ketamine/saline control and 2 days after. The resulting dataset in FIG. 5 demonstrates that the correlation among neurosphere units of NNet is decreased in ketamine treated preparations. The graphs show dF/F traces of different neurospheres from the same network. On the right in FIG. 5, heat maps show pairwise-correlation among neurospheres. As evident, NNet activity correlation increases with time in control conditions, but decreases in the ketamine model. These results are consistent with data published from Schizophrenia studies in mice, which indicates that the NNet approach is useful for modeling complex brain disorders. Example 6: Modeling epileptic seizures with NNet.

4-AP, a commonly used seizure inducing compound, was injected in a specific neurosphere of an NNet preparation. In FIG. 6, the first graph on the top shows the typical high seizure peaks produced by 4-AP local injection. As shown in the magnified view (top row, third) these peaks are maintained during the time. However, the addition of picrotoxin (GABA receptor antagonist) reduces the inhibition resulting in increased frequency of number of events. In FIG. 6 the colored maps (bottom) show the spatial progression of the seizure waves in three different bouts (labeled as Epi. Events #1, #2, and #3). The directionality of seizure spread is maintained constant from bottom-left to top-right. However, when picrotoxin is added, the directionality becomes random (see bottom of FIG. 6). These results are consistent with the data published from Epilepsy studies on brain mice, which indicates that the NNet approach is useful for modeling complex brain disorders.

Example 6: Drug screening.

In a typical drug screening experiment, the NNet cultures (as described elsewhere), either derived from brain cells taken from a disease model or differentiated from iPSC cells from a patient, will be exposed to a candidate drug molecule. In parallel, a control network derived as above will be exposed to the same procedure but without the drug molecule. In addition, optionally when available, the NNet cultures would be prepared from mammal 2 with no known diseases. Note that, with the accumulation of data, we may not always need to use sample from mammal 2 as we will have enough

documentation of normal behavior of such preparation.

These cultures will be subjected to high-speed live imaging in regular interval (e.g. every 3 hours) for several days (e.g. 10 days) to capture the activities of all brain cells, and their evolution over time. At the end of the live recording experiments, these cultures will be fixed using chemical fixatives (e.g. paraformaldehyde), and will be stained with antibodies and other reagents (e.g., antisense RNA) to identify molecular identity/type of all neurons. The live recording image datasets will be analyzed to extract activity patterns of individual neurons, which will be further quantified with a multitude of descriptors, including the activity pattern motifs, the development of local (i.e. within units) and global synchrony over time. These descriptors will be compared across the preparations to assess the level of recovery achieved by the tested candidate drug molecule. Such process will be repeated for a large number of potential drug candidates, either manually or by an automated process, one implementation of which may utilize microfluidics based devices for automating the growing of the culture, and the delivery of specific drug candidates, while recording with a microscope device.

Note that all experiments will be performed under strict control of environment parameters including temperature and humidity.

Although in embodiments, a mold of agarose is described, it is understood that other types of hydrogel or other non-adhesive polymers may be substituted therefore to generate additional embodiments.

All references cited herein are incorporated by reference in their entirety. While the above disclosure has been described with reference to exemplary embodiments, those of ordinary skill in the art will understand that various changes in form and details may be made without departing from the spirit and scope of the present invention as defined by the following claims.