Methods and compositions for increasing neuronal activity and synaptic plasticity

Abstract

Disclosed are compositions and methods for inducing tonic release of glutamate and increasing synapse formation. For example, disclosed is a method for inducing tonic release of glutamate, comprising administering vandefitemcel to a brain region of a subject. The vandefitemcel can be a cell derived from mesenchymal stem cells (MSCs) transiently transfected with a polynucleotide encoding a Notch intracellular domain (NICD). Further, for example, disclosed is a method of increasing synapse formation in neurons. The method can comprise capturing a functional magnetic resonance imaging (fMRI) scan of a brain region of a subject, selecting a brain region showing neuronal activity based on the fMRI scan, and administering vandefitemcel to the selected brain region comprising neurons.

Description

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 63 / 626,785, filed January 30, 2024, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure relates generally to the field of cell therapy, and more specifically to methods and compositions for increasing neuronal activity and synaptic plasticity in subjects with corresponding needs.

[0003] background Over the past few decades, mesenchymal stem cell-based therapies have emerged as a novel strategy for treating neurological disorders and injuries such as stroke, traumatic brain injury (TBI), amyotrophic lateral sclerosis (ALS), and spinal cord injury (SCI) [1, 2]. These neurological disorders or injuries often involve impairment of certain cognitive functions due to disruptions in neuronal communication. For neuronal communication to be restored and for functional recovery to be facilitated, there must be an increase in neuronal activity and synaptic plasticity, which allows for the repair of certain neural pathways or the creation of new ones.

[0004] MSCs are low in immunogenicity and can be isolated from various adult and birth tissues and can be cultured with strong expansion capacity[3]. Vandefitemcel, also known as SB623 cells, is a type of MSC derived from human bone marrow. Recent studies have shown that intracerebral implantation of vandefitemcel or SB623 cells is safe and can improve motor function in patients[4, 5]. However, little is known about the electrophysiological effects of MSCs on neurons (and particularly human neurons) after implantation or transplantation.

[0005] While neurons used for research can come from a variety of sources, one regenerative source of human neurons that does not require invasive biopsies of human central nervous system (CNS) tissue is human induced pluripotent stem cells (iPSCs). Human iPSCs can differentiate into a range of somatic cell types, including neuronal types such as glutamatergic neurons and GABAergic neurons [6].

[0006] In addition, multi-electrode arrays (MEAs) have been used in the past to measure the electrophysiology of neurons [7]. MEAs typically consist of specialized multi-well culture plates in which small electrodes are embedded at the bottom of each well. Extracellular spontaneous action potentials are recorded from neurons cultured directly on top of the recording electrodes.

[0007] Therefore, there is a need for safe and effective cell-based therapies to increase neuronal activity and synaptic plasticity. Such therapies should be testable on human iPSC-derived neurons using state-of-the-art MEA measurement systems.

[0008] Overview In some aspects, a method for inducing, triggering, or causing tonic release of glutamate in a subject is disclosed. This method involves administering vandefitemcel to a brain region of the subject. The vandefitemcel may be derived from mesenchymal stem cells (MSCs) transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD).

[0009] In some respects, administering vandefitemcel to brain regions of subjects also includes administering vandefitemcel via intracerebral implantation.

[0010] In some respects, when implanted via intrabrain implantation, vandefitemcel can release glutamate to neurons in the brain in a tonic manner.

[0011] In some respects, administering vandefitemcel to brain regions of subjects also includes injecting vandefitemcel at multiple sites within the brain region.

[0012] In some respects, the administration of vandefitemcel to brain regions of subjects also includes stereotactic administration of vandefitemcel via a drill hole in the subject's skull.

[0013] In some respects, the brain region is the subject's forebrain, including at least one of the subject's cerebrum, thalamus, and hypothalamus.

[0014] In some respects, brain regions can be sites of injury or disease.

[0015] In some respects, the brain region could be the subject's hippocampus.

[0016] In some respects, administering vandefitemcel to brain regions of subjects also includes administering vandefitemcel via parenteral administration.

[0017] In some cases, vandefitemcel can be suspended in a sterile isotonic crystalloid solution.

[0018] In some respects, the administration of vandefitemcel to brain regions of subjects also includes the administration of between approximately 1 million and 10 million cells.

[0019] In some respects, vandefitemcel can be prepared by methods including: providing a culture of MSCs; contacting the MSC culture with a polynucleotide encoding NICD, wherein the polynucleotide does not encode the full-length Notch protein; selecting cells containing the polynucleotide; and further culturing the selected cells in the absence of selection for the polynucleotide.

[0020] In some respects, MSCs can be cells derived from human bone marrow.

[0021] In some aspects, the method may also include capturing functional magnetic resonance imaging (fMRI) scans of brain regions of the subject, selecting a brain region displaying neuronal activity based on the fMRI scans, and applying vandefitemcel to said brain region.

[0022] In some aspects, a method for increasing synapse formation in neurons is disclosed. This method may include applying vandefitemcel to a neuronal-containing brain region of a subject. The vandefitemcel may be a cell passaged from mesenchymal stem cells (MSCs) transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD).

[0023] In some respects, the administration of vandefitemcel also includes administration via intracerebral implantation.

[0024] In some respects, after vandefitemcel is implanted via intrabrain implantation, vandefitemcel can release glutamate to neurons in the subject's brain in a tonic manner.

[0025] In some respects, the administration of vandefitemcel also includes injecting vandefitemcel at multiple sites within the brain region.

[0026] In some respects, the administration of vandefitemcel also includes stereotactic administration of vandefitemcel via a hole drilled in the subject's skull.

[0027] In some respects, the brain region is the subject's forebrain, including at least one of the subject's cerebrum, thalamus, and hypothalamus.

[0028] In some respects, brain regions can be sites of injury or disease.

[0029] In some respects, the brain region could be the subject's hippocampus.

[0030] In some respects, the administration of vandefitemcel may also include administration via parenteral administration.

[0031] In some cases, vandefitemcel can be suspended in a sterile isotonic crystalloid solution.

[0032] In some respects, the application of vandefitemcel may also include the application of vandefitemcel between approximately 1 million and 10 million cells.

[0033] In some respects, vandefitemcel can be prepared by methods including: providing a culture of MSCs; contacting the MSC culture with a polynucleotide encoding NICD, wherein the polynucleotide does not encode the full-length Notch protein; selecting cells containing the polynucleotide; and further culturing the selected cells in the absence of selection for the polynucleotide.

[0034] In some respects, MSCs can be cells derived from human bone marrow.

[0035] In some aspects, the method may also include capturing functional magnetic resonance imaging (fMRI) scans of brain regions of the subject, selecting a brain region displaying neuronal activity based on the fMRI scans, and applying vandefitemcel to said brain region.

[0036] In some aspects, a composition for increasing synapse formation in neurons is disclosed. The composition may contain vandefitemcel in an amount between approximately 1 million and 10 million cells and one or more pharmaceutically acceptable excipients. Vandefitemcel can be produced by modifying mesenchymal stem cells derived from human bone marrow.

[0037] In some respects, vandefitemcel can be prepared by a process comprising: providing a culture of mesenchymal stem cells (MSCs); contacting the MSC culture with a polynucleotide encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full-length Notch protein; selecting cells containing the polynucleotide; and further culturing the selected cells in the absence of selection for the polynucleotide.

[0038] In some respects, MSCs can be cells derived from human bone marrow.

[0039] In some respects, MSCs can be transiently transfected using plasmid vectors containing polynucleotides encoding NICD.

[0040] In some respects, one or more pharmaceutically acceptable excipients may include at least one of buffers, proteins, stabilizers, and preservatives.

[0041] In some cases, vandefitemcel can be suspended in a sterile isotonic crystalloid solution.

[0042] In some respects, one or more pharmaceutically acceptable excipients may include carriers or diluents.

[0043] In some aspects, a method for inducing, triggering, or causing glutamate release in a subject is disclosed. This method may include capturing a functional magnetic resonance imaging (fMRI) scan of a brain region of the subject, selecting brain regions displaying neuronal activity based on the fMRI scan, and administering vandefitemcel to the selected brain region. Vandefitemcel may be derived from mesenchymal stem cells (MSCs) transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD). Upon administration via intrabrain implantation, vandefitemcel releases glutamate.

[0044] In some aspects, a method for increasing synapse formation in neurons is disclosed. This method may include capturing functional magnetic resonance imaging (fMRI) scans of brain regions of a subject, selecting brain regions displaying neuronal activity based on the fMRI scans, and administering vandefitemcel to the selected brain regions containing neurons. Vandefitemcel may be derived from mesenchymal stem cells (MSCs) transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD). Upon administration via intrabrain implantation, vandefitemcel releases glutamate into the neurons. Brief description of the attached diagram Figures 1A-1D This is a graph showing the electrophysiological activity and viability of individual cultures of vandefitemcel and iFbN over an eight-week period using a multi-electrode array (MEA) system.

[0046] Figure 1E This is a raster plot showing the electrophysiological activity and network burst development of iFbN cell cultures at different cell densities at four and eight weeks.

[0047] Figure 2A This is a reproduction of the immunocytochemical staining of a co-culture of vandefitemcel and iPSC-derived forebrain neurons (iFbN) cells.

[0048] Figure 2BThis figure illustrates the presence of vandefitemcel in a co-culture of vandefitemcel and iFbN cells. The presence of vandefitemcel is confirmed by the increase in resistance measured between the cells and the MEA electrode.

[0049] Figure 2C This is a bar graph illustrating the increased neuronal excitability detected in the iFbN and vandefitemcel co-culture compared to the iFbN culture alone, based on the difference in the number of observed spikes after two weeks of co-culture.

[0050] Figure 2D This is a bar graph illustrating that adding vandefitemcel to iFbN cells during four weeks of co-culture increased the number of network bursts in a cell density-dependent manner.

[0051] Figure 2E This figure illustrates the increase in glial fibrillary acidic protein (GFAP) cells in the co-culture of iFbN cells and vandefitemcel compared to the iFbN control culture. Figure 2E It also indicates that the 5-ethynyl-2'-deoxyuridine (EdU) population is comparable for both the iFbN and vandefitemcel co-cultures and the iFbN control culture.

[0052] Figures 3A-3B The bar graph shows that after adding vandefitemcel to iFbN cells, the co-culture of vandefitemcel and iFbN produced significantly more spikes and network bursts than the iFbN cell culture alone. Furthermore, Figures 3A-3B The study showed that co-cultures of vandefitemcel conditioned medium and iFbN produced more spikes and network bursts than iFbN cells alone. Furthermore, co-cultures of human astrocytes and iFbN also produced more spikes and network bursts than iFbN cells alone.

[0053] Figure 3C This is a raster diagram showing the electrophysiological activity and network burst development of individual iFbN cells, as well as co-cultures of vandefitemcel and iFbN, co-cultures of vandefitemcel in conditioned medium and iFbN, and co-cultures of human astrocytes and iFbN, across MEA electrodes for four weeks.

[0054] Figure 4A This is a reproduction of various immunocytochemical (ICC) stainings of iFbN cultures at 30 days of culture maturation.

[0055] Figure 4B This is a bar graph illustrating that on day 3 of co-culture, vandefitemcel promoted high levels of spike activity in glutamatergic neurons compared to spike activity in isolated glutamatergic neurons or in co-cultures of glutamatergic neurons with human astrocytes.

[0056] Figure 4C This diagram illustrates that vandefitemcel-induced spike activity in glutamatergic neurons continues to increase for two weeks, while human astrocytes (hA) slightly promote spike activity in glutamatergic neurons starting from the third week.

[0057] Figure 4D This diagram illustrates that vandefitemcel induces significant network burst activity in the first week of co-culturing with glutamatergic neurons, while glutamatergic neurons (GluN+hA) co-cultured with human astrocytes do not show network burst activity until the fourth week.

[0058] Figure 4E It is a raster diagram illustrating the electrophysiological activity of co-cultures of glutamatergic neurons and vandefitemcel over time, as well as the progressive network burst development across electrodes in the MEA system.

[0059] Figure 4F and Figure 4G This is a graph illustrating that vandefitemcel promoted spikes and network bursts of GABAergic neurons in weeks 2 and 4 of co-culture compared to individual GABAergic neurons or co-cultures of GABAergic neurons and human astrocytes.

[0060] Figure 4H This is a raster diagram showing the electrophysiological activity and network burst development of co-cultures of GABAergic neurons and vandefitemcel over time, as well as the progressive network burst development across electrodes on the MEA system.

[0061] Figure 5A This indicates that after two days of culture, the increase in glutamate levels in conditioned medium (CM) was cell density-dependent in both vandefitemcel and human astrocyte (hA) cultures, but the glutamate level in vandefitemcel CM was approximately 10 times higher than that in hA CM (bar graph).

[0062] Figure 5B The graph shows that after 5 days of culture, the level of glutamate in the CM of vandefitemcel increased by 4.9 times, while the level of glutamate in the CM of hA increased by 2.7 times.

[0063] Figure 5C The bar graphs show that after incubation with 5 μM and 50 μM glutaminase inhibitor (CB-839), the level of glutamate in the CM of vandefitemcel was reduced by 1.5-fold and 2.0-fold, respectively, compared with the control of vandefitemcel incubated with dimethyl sulfoxide (DMSO).

[0064] Figure 5D This is a bar graph illustrating that incubation with CB-839 does not alter the viability of vandefitemcel.

[0065] Figure 5E and Figure 5F The bar graph shows that, compared with the vandefitemcel control culture incubated with DMSO, after one week of co-culture, the inhibition of glutaminase in vandefitemcel significantly reduced the spike activity and network bursts in glutamatergic neurons and in the vandefitemcel co-culture.

[0066] Figure 5G and Figure 5H This is a bar graph showing the reduction in the number of spikes and network bursts that would normally be induced by vandefitemcel on glutamatergic neurons.

[0067] Figure 5I This is a schematic diagram showing that vandefitemcel promotes increased neuronal activity and synaptic plasticity through tonic glutamate release.

[0068] Detailed Explanation For the purposes of this disclosure, the following terms are defined as follows.

[0069] definition The terms “administration” and “administering” refer to the physical introduction of a composition containing a therapeutic agent into a subject using any of the various methods and delivery systems known to those skilled in the art. For example, the route of administration of vandefitemcel may include intracerebral, intrathecal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase “parenteral administration” refers to a method of administration other than enteral and topical administration, typically by injection and infusion. Administration may also be performed, for example, once, more than once, and / or over one or more extended time periods, and may be a therapeutically effective dose or a subtherapeutic dose.

[0070] The terms “implantation” and “transplantation” are used to refer to the introduction of exogenous cells (e.g., vandefitemcel or SB623 cells) into a subject or patient. The exogenous cells can be autologous (i.e., obtained from the subject) or allogeneic (i.e., obtained from an individual other than the subject).

[0071] An "isotonic crystalloid solution" is a solution containing water-soluble electrolytes. Such a solution contains the same amount of electrolytes as blood plasma.

[0072] "Mesenchymal cells" refers to cells of mesenchymal tissue (e.g., chondrocytes, osteoblasts, osteocytes, adipocytes) and their precursors, and includes, for example, fibroblasts (e.g., human foreskin fibroblasts), MSCs (as defined herein), and cells derived from MSCs, such as vandefitemcel, as defined herein.

[0073] “MSC” (“mesenchymal stem cells”) refers to non-hematopoietic pluripotent cells that adhere to the bone marrow. These cells are referred to by various names, such as mesenchymal stem cells, mesenchymal stromal cells, bone marrow adherent stromal cells, bone marrow adherent stem cells, and bone marrow stromal cells. MSCs can also be obtained from, for example, umbilical cord blood, adipose tissue, dental pulp, Wharton's jelly, and various types of connective tissue. MSCs can be obtained by selecting (e.g., by growing in a culture) adherent cells (i.e., cells that adhere to tissue culture plastic) from the bone marrow. In order to obtain a population of MSCs with a sufficient number of cells for treatment, the population of adherent cells is expanded in a culture after selection for adhesion. The expansion in the culture also enriches MSCs because contaminating cells (e.g., monocytes) do not proliferate under culture conditions. Exemplary disclosures of MSCs are provided in U.S. Patent Publication No. 2003 / 0003090 and reference [8]. Methods for isolating and purifying MSCs can be found, for example, in U.S. Patent No. 5,486,359 and references [9, 10]. Human MSCs are commercially available (e.g., BioWhitaker, Walkersville, Md.) or can be obtained from a donor by, for example, bone marrow aspiration, followed by culturing and selecting adherent bone marrow cells. See, for example, WO 2005 / 100552. MSCs can also be isolated from umbilical cord blood [11, 12]. Other sources of MSCs include, for example, adipose tissue, dental pulp, and Wharton's glume.

[0074] "Network burst" refers to the collective or synchronous / near-synchronous behavior of neurons, which includes relatively rapid peaks or alternating periods of peak activity followed by inactivity. It is also known as swarm burst.

[0075] A "neuronal spike" or "spiking point" refers to an action potential or brief electrical impulse that can be captured or recorded when a neuron fires. Spikes can be acquired by electrodes such as those in a microelectrode array.

[0076] "Neurodegenerative disorder," "neurological disorder," or "neurological disease" refers to a disorder or disease in which cells of the central nervous system (CNS) cease to function, die, or are damaged in some way. These types of diseases or disorders may include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), traumatic brain injury (TBI), ischemic stroke, hemorrhagic stroke, chronic stroke, or spinal cord injury.

[0077] Notch proteins (e.g., Notch 1 protein) are transmembrane receptors found in all metazoans that influence cell differentiation through intracellular signaling. Contact between the Notch extracellular domain (e.g., the extracellular domain of Notch 1 protein) and Notch ligands (e.g., Delta, Serrate, Jagged) leads to two proteolytic cleavages of the Notch protein, the second of which is catalyzed by γ-secretase and releases the Notch intracellular domain (NICD) into the cytoplasm. In mouse Notch proteins, this cleavage occurs between amino acids gly1743 and val1744. The NICD translocates to the nucleus, where it acts as a transcription factor, recruiting additional transcriptional regulatory proteins (e.g., MAM, histone acetyltransferases) to alleviate transcriptional repression of various target genes (e.g., Hes 1). Further details and information regarding Notch signaling can be found in references [13, 14, 15].

[0078] The term "pharmaceuticalally acceptable excipient" refers to an excipient used to administer a pharmaceutical preparation. These "excipients" refer to any substance other than the active pharmaceutical ingredient. Examples of pharmaceutically acceptable excipients may include, but are not limited to, buffers, proteins, carbohydrates, oleochemicals, petrochemicals, stabilizers, preservatives, fillers, diluents, binders, viscosity agents, coatings, disintegrants, colorants, and lubricants.

[0079] The terms “vandefitemcel,” “vandefitemcel cells,” “SB623,” and “SB623 cells” refer to cell populations obtained by transiently expressing a foreign Notch intracellular domain (NICD) in MSCs. For example, a vandefitemcel or SB623 cell population can be obtained by transiently transfecting MSCs with a plasmid vector containing a sequence encoding a NICD (e.g., from the human Notch 1 protein) but not the full-length Notch protein, followed by selection (e.g., with G418). The selected cells can be further cultured in standard medium, optionally supplemented with serum, without any added growth factors or differentiation factors (except those that may be present in serum, if serum is present in the medium). Vandefitemcel can be derived from human (allogeneic) bone marrow MSCs by transiently transfecting them with a NICD (e.g., the human Notch 1 intracellular domain (NICD1)), followed by selection and subsequent expansion. This process produces a cell population distinct from the parental MSCs [5,6,7]. vandefitemcel or SB623 cells are also known as progeny of NICD transiently transfected MSCs (“DNTT-MSC”).

[0080] The term "transient transfection" refers to a method of introducing foreign DNA or RNA into cells to temporarily express the introduced gene. Transient transfection affects short-term gene expression, lasting from a few hours to a few days.

[0081] The terms "tonically released glutamate" and "tonically released glutamate" refer to the non-vesicular release of glutamate from neurons or glial cells into the extracellular space. Tonifying glutamate release can help maintain or regulate baseline neuronal activity in subjects and help maintain or regulate synaptic plasticity.

[0082] "Therapeuticly effective" means the amount of medication required to provide meaningful patient benefit or promote disease remission, demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of damage or disability caused by disease suffering. A variety of methods known to skilled practitioners (e.g., those in the field of neurology) can be used to evaluate the therapeutically effective amount of medication.

[0083] Preparation of vandefitemcel The applied vandefitemcel is an allogeneic cell derived from mesenchymal stem cells transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD). These cells can be prepared by a method comprising: providing a culture of mesenchymal stem cells; contacting the culture of mesenchymal stem cells with a polynucleotide encoding the NICD (wherein the polynucleotide does not encode the full-length Notch protein); selecting cells containing the polynucleotide; and further culturing the selected cells in the absence of selection targeting the polynucleotide. The mesenchymal stem cells may be human bone marrow-derived cells.

[0084] As discussed above, vandefitemcel can be obtained from bone marrow adhesion stromal cells (also known as MSCs) by transiently expressing the intracellular domain of the Notch protein in MSCs. Transient expression of the Notch intracellular domain (e.g., NICD from the human Notch 1 protein) in MSCs is sufficient to convert the MSC population into a vandefitemcel population. No further treatment with growth factors and / or differentiation factors is required. Therefore, the MSC population can be converted into a vandefitemcel population by transiently transfecting MSCs with a plasmid vector containing a sequence encoding NICD (but not the full-length Notch protein), selecting cells containing the vector, and further culturing the selected cells in serum-containing medium without exposure to additional growth factors and / or differentiation factors. See, for example, U.S. Patent No. 7,682,825; U.S. Patent No. 8,945,919; and WO 2009 / 023251; the contents of which are incorporated herein by reference in their entirety for the purpose of describing the isolation of mesenchymal stem cells and the conversion of mesenchymal stem cells into vandefitemcel (also referred to in those documents as “neural progenitor cells” and “neural regeneration cells”).

[0085] In this disclosure, any polynucleotide encoding the Notch intracellular domain (e.g., plasmid vector) can be used, and any method for selecting and enriching transfected cells can be used. For example, MSCs can be transfected with a vector containing a sequence encoding the Notch intracellular domain (e.g., the human Notch 1 intracellular domain) and also containing a sequence encoding a selection marker (e.g., resistance; such as resistance to G418). In some cases, two plasmid vectors (one containing a sequence encoding the Notch intracellular domain and the other containing a sequence encoding a resistance marker) can be used to transfect MSCs. In these cases, after transfecting cell cultures with one or more vectors, selection is achieved by adding a selection agent (e.g., G418) to the cell culture in an amount sufficient to kill cells that do not contain the vector but not cells that do contain the vector. There is no selection requirement to remove the selection agent or reduce its concentration to a level that does not kill cells that do not contain the vector. After selection (e.g., for seven days), the selection agent can be removed, and cells can be further cultured in a serum-containing medium (e.g., for two passages).

[0086] Depending on the nature of the selection marker and / or the concentration of the selector used, not every cell lacking a carrier encoding the selection marker may be killed during the selection process. For example, the selector may inhibit the growth of cells that do not contain the selection marker, and the cells may recover and resume growth after the selector is removed.

[0087] Therefore, the preparation of vandefitemcel involves the transient expression of exogenous Notch intracellular domains in MSCs. For this purpose, MSCs can be transfected with plasmid vectors containing sequences encoding Notch intracellular domains (e.g., human Notch 1 intracellular domains) that do not encode the full-length Notch protein. All of these sequences are known and readily available to those skilled in the art

[14] .

[0088] Similar information is available for Notch proteins and nucleic acids from other species, including rats, Xenopus, fruit flies, and humans [16, 17]. Additional information can also be found in NCBI reference sequence NM_017167, SwissProtP46531, SwissProtQ01705, and GenBank CAB40733. The aforementioned references are incorporated in their entirety by reference for the purpose of disclosing the amino acid sequences of the full-length Notch protein and the amino acid sequences of the Notch intracellular domain from many different species.

[0089] In some cases, vandefitemcel can be prepared by introducing nucleic acids containing sequences encoding the Notch intracellular domain into MSCs, thereby preventing the MSCs from expressing exogenous Notch extracellular domains. This can be achieved, for example, by transfecting MSCs with a plasmid vector containing a sequence encoding the Notch intracellular domain, wherein the sequence does not encode the full-length Notch protein.

[0090] Further details regarding the preparation of vandefitemcel or SB623 cells and methods for preparing cells having properties similar to those of vandefitemcel or SB623 cells that can be used with the methods disclosed herein can be found in references

[18] and U.S. Patent Nos. 7,682,825, 8,945,919, and 9,441,199, the contents of which are incorporated herein by reference in their entirety for the purpose of describing alternative methods for preparing vandefitemcel and providing methods for preparing cells having properties similar to those of vandefitemcel.

[0091] Vandefitemcel cells used in the experiments disclosed herein were stored in liquid nitrogen. For co-cultures, unless otherwise specified, vandefitemcel cells were thawed, washed, and plated directly. For glutaminase inhibition assays, vandefitemcel (or SB623) cells were thawed and cultured in minimally essential medium-α (α-MEM) (from CORNING, Corning, NY) supplemented with 10% fetal bovine serum (FBS). In co-cultures with neurons or for glutamate assays, vandefitemcel (or SB623) cells were maintained in BrainPhys™ medium supplemented with NeuroCult™ SM1 neuronal supplement. To assess the level of glutamate released into the conditioned medium by vandefitemcel cells, Gibco GlutaMAX™ (100X; 200 mM L-alanyl-L-glutamine from Thermo Fisher Scientific, Waltham, MA) (final concentration 2 mM) was added to vandefitemcel cells cultured in BrainPhys™ medium supplemented with NeuroCult™ SM1 neuron complement, and glutamate levels were assessed after two days.

[0092] transfection Methods for introducing exogenous DNA into cells (i.e., transfection) and selecting transfected cells are known in the art [19,20].

[0093] Preparation of iPSC-derived forebrain neurons (iFbN) Human augmentative iPSCs (e.g., Gibco™ A18945 hiPSCs from Thermo Fisher Scientific, Waltham, MA) were grown in six-well tissue culture plates pre-coated with Geltrex™ hESC-qualified (from Thermo Fisher Scientific) using the StemFlex™ medium kit (from Thermo Fisher Scientific) and passaged using ReLeSR™ (from Stem Cells Technologies, Vancouver, Canada). To generate forebrain neurons, the iPSCs were differentiated into neural progenitor cells using the STEMdiff™ forebrain neuron differentiation kit (from Stem Cells Technologies, Vancouver, Canada). Following the single-cell dissociation procedure, iPSCs were seeded in six-well tissue culture plates pre-coated with 15 μg / mL poly-L-ornithine (from Sigma-Aldrich, St. Louis, MO) and 20 μg / mL laminin (from Sigma-Aldrich, St. Louis, MO), and differentiated using a monolayer culture system in StemDiff™ Neural Induction Medium plus SMADi (both from Stem Cells Technologies, Vancouver, Canada), followed by a neural induction procedure (Stem Cells Technologies, Vancouver, Canada). Neural progenitor cells were passaged into new six-well tissue culture plates pre-coated with 15 μg / mL poly-L-ornithine and 20 μg / mL laminin, and then differentiated into forebrain neurons for one week using the STEMdiff™ Forebrain Neuron Differentiation Kit. After differentiation, forebrain neurons were matured for two weeks using the STEMdiff™ Forebrain Neuron Maturation Kit (from Stem Cells Technologies, Vancouver, Canada). The forebrain neuron cultures were then maintained in BrainPhys™ medium supplemented with NeuroCult™ SM1 neuron supplement (from Stem Cells Technologies, Vancouver, Canada). All cells were grown in a humid, sterile environment at 37°C and 5% CO2.

[0094] Preparation of human glutamatergic neurons, human GABAergic neurons and human astrocytes Human glutamatergic neurons (e.g., iCell GlutaNeurons™) and human GABAergic neurons (e.g., iCell GABAneurons™) were obtained from FUJIFILM Cellular Dynamics Inc. (Madison, WI) and cultured directly in BrainPhys™ medium supplemented with NeuroCult™ SM1 neuron supplement on multi-electrode array (MEA) plates / wells pre-coated with 15 μg / mL poly-L-ornithine (from Sigma-Aldrich, St. Louis, MO) and 20 μg / mL laminin (from Sigma-Aldrich, St. Louis, MO). Human fetal-derived astrocytes (from ScienceCell Research Laboratories Carlsbad, CA) were seeded on T-75 flasks pre-coated with 10 μg / mL poly-L-lysine (from Sigma-Aldrich, St. Louis, MO) and expanded in astrocyte medium supplemented with 2% fetal bovine serum (FBS) and 1% astrocyte supplement (both from Science Cell Research Laboratories). Human astrocytes were held in BrainPhys™ medium supplemented with NeuroCult™ SM1 neuron supplement before co-culturing with neurons or for use in glutamate assays.

[0095] Multi-electrode array (MEA) system Treat CytoView MEA 24 wells / plates (from Axion Biosystems, Atlanta, GA) with 30–50 μl of 15 μg / mL poly-L-ornithine at 37°C and 5% CO2 for 2 hours or overnight. Wash wells twice with phosphate-buffered saline (PBS) and once with Dulbecco modified Eagle medium / nutrient mix F-12 and 4-(2-hydroxyethyl)piperazine-1-ethane-sulfonic acid (DMEM / F12-HEPES) (from Stem Cells Technologies, Vancouver, Canada). Coat wells with 30–50 μl of 20 μg / mL laminin (from Sigma-Aldrich, St. Louis, MO) at 37°C and 5% CO2 for 2 hours or overnight. Remove laminin and immediately seed neurons, incubating at 37°C and 5% CO2 for 30–45 minutes. Then add fresh medium to reach a volume of 400 μl per well. Replace half the culture medium volume every two to three days. Forebrain neurons (iFbN) are grown at a density of 3 x 10⁻⁶ cells per well. 4 Or 5 x 10 4 Cells were cultured for two weeks in BrainPhys™ (from Stem Cells Technologies, Vancouver, Canada) supplemented with a mature supplement. After two weeks, the medium was replaced with BrainPhys™ medium supplemented with NeuroCult™ SM1 neuronal supplement. To co-culture vandefitemcel cells or human astrocytes with iPSC-derived forebrain neurons (iFbNs), the medium was transferred to 24-well plates, leaving approximately 10–20 μL in the inner wells of the MEA plate. Vandefitemcel (or SB623) cells or astrocytes were added to 10–20 μL of cell solution and incubated at 37°C and 5% CO2 for 45 minutes. After incubation for 30–45 minutes, a 1:1 mixture of fresh medium and stored conditioned medium was added to reach a volume of 400 μL per well. Half the volume of medium was replaced every three or four days.

[0096] Glutamate neurons or GABAergic neurons were divided into 9 x 10 groups. 4 1 cell per well or 1 x 10 cells per well 5 Cells were seeded at 25 μL volumes in BrainPhys™ medium supplemented with NeuroCult™ SM1 neuronal complement and incubated at 37°C and 5% CO2 for 30–45 minutes. For co-culture, 10–20 μL of 1.5 x 10⁻⁶ cells per well was added. 4To co-culture vandefitemcel (or SB623) cells or astrocytes, first saturate the cells with a cell solution and then incubate at 37°C and 5% CO2 for 45 minutes. Add fresh medium to reach a volume of 400 μL per well and incubate the cells at 37°C and 5% CO2. Replace half the volume of medium every three or four days.

[0097] To isolate the presynaptic activity of iFbN on a MEA after 50 days of culture, 50 μM of the broad-spectrum glutamate receptor antagonist kynurenic acid (from R&D Systems, Minneapolis, MN) was added 90 seconds into the recording. Spike activity was evaluated to detect the blockade of glutamate receptor activity.

[0098] MEA recordings were performed at 37°C and 5% CO2 using a Maestro Edge (from Axion Biosystems, Atlanta, GA). The medium was changed at least two hours prior to recording. The CO2 level was adjusted to 5% prior to recording. AxISNavigator 3.6.2 from Axion Biosystems with the configuration module Neural Real-Time; Spontaneous + Viability was used, with each recording lasting five minutes and a minimum spike rate of 5 spikes / min for the active electrode.

[0099] Flow cytometry iPSC-derived forebrain neuron cultures were dissociated into single-cell suspensions from the multi-electrode wells. For this purpose, the cultures were washed once with phosphate-buffered saline (PBS) (from Thermo Fisher Scientific, Waltham, MA) and incubated for 5 min at 37°C and 5% CO2 with TrypLE™ express (from Thermo Fisher Scientific, Waltham, MA). Cells were recovered in BrainPhys™ medium, washed 2x, and then fixed for 30 min at room temperature with eBioscience™ IC fixation buffer in 500 μL PBS (from Thermo Fisher Scientific, Waltham, MA). After fixation, the cells were washed twice at 300 g for 5 min each time with 2 mL eBioscience™ permeabilization buffer (from Thermo Fisher Scientific, Waltham, MA). Cells were incubated for 15 minutes at room temperature using an orbital shaker with a blocking solution containing 5% normal goat serum (from Jackson ImmunoResearch Inc., West Grove, PA) + 2% bovine serum albumin (BSA) in eBioscience™ permeabilization buffer, protected from light. Without washing the cells, conjugated antibodies were added directly to the cells to detect intracellular antigens, and the cells were incubated for 30 minutes at room temperature using an orbital shaker, protected from light. The conjugated antibodies used were the REA control antibody REAfinity™ and the GFAP antibody REAfinity™ (both from Miltenyi Biotec, San Jose, CA). Cells were washed twice with eBioscience™ permeabilization buffer at 300 g for 5 minutes each time and resuspended in eBioscience™ flow cytometry staining buffer (from Thermo Fisher Scientific, Waltham, MA). For proliferation assays, the Click-iT® 5-ethynyl-2'-deoxyuridine (EdU) flow cytometry assay kit (from Thermo Fisher Scientific, Waltham, MA) was used. EdU was added for up to 24 hours. Flow cytometry was performed using a MACSQuant® Analyzer 10 and data were analyzed using MACSQuantify™ (both from Miltenyi Biotec, San Jose, CA).

[0100] Immunocytochemical staining iFbN and vandefitemcel (or SB623) cells were co-cultured on Nunc™ Lab-Tek™ II CC2 slides pre-coated with poly-L-ornithine and laminin. Cells were washed with PBS (from Thermo Fisher Scientific, Waltham, MA) and then incubated with 4% paraformaldehyde for 15 min at room temperature. For staining, samples were blocked for 30 min at room temperature with 5% normal goat serum (from Jackson ImmunoResearch Laboratories Inc., West Grove, PA) in PBS containing a nonionic surfactant (e.g., 0.3% Triton X-100 from Sigma-Aldrich). The primary antibodies used were rabbit anti-GFP (1:200; from Abcam, Fremont, CA), rabbit anti-GFAP (1:1000; from Agilent DAKO), rabbit anti-Olig2 (1:100; from Cell Signaling, Danvers, MA), chicken anti-MAP2 (1:1000; from Abcam, Fremont, CA), rabbit anti-synaptophysin (1:500; from Abcam, Fremont, CA), mouse anti-VGLUT1 (1:1000), and rabbit anti-GABA (1:100; from Sigma-Aldrich, St. Louis, MO). Cells were washed three times with PBS containing a nonionic surfactant (e.g., 0.3% Triton X-100 from Sigma-Aldrich, St. Louis, MO) and incubated overnight at 4°C with the corresponding secondary antibodies. The secondary antibodies used were Alexa Fluor 488-conjugated goat anti-rabbit (1:1000; Invitrogen, Waltham, MA), Alexa Fluor™ 546-conjugated goat anti-mouse (1:1000; from Invitrogen, Waltham, MA), and Alexa Fluor™ 647-conjugated goat anti-chicken (1:1000; from Invitrogen, Waltham, MA). The slides were then washed three times with PBS containing a nonionic surfactant (e.g., 0.3% Triton X-100) for 5 minutes each time. The slides were mounted with Fluoromount-G™ mounting medium containing 4',6-diamidinyl-2-phenylindole (DAPI) (from Thermo Fisher Scientific, Waltham, MA).Fluorescence images were recorded using a digital camera attached to a fluorescence microscope (Leica DMi8, Wetzlar, Germany). Image processing was performed using Fiji-ImageJ software (National Institute of Health; NIH). Antibody specificity and sample background were tested by using only secondary antibody on each stained section.

[0101] Determination of glutamate concentration The glutamate concentration in the conditioned medium was determined using a glutamate assay kit from Sigma-Aldrich (St. Louis, MO) and according to the manufacturer's instructions. Cells were seeded at different cell densities in BrainPhys™ medium supplemented with NeuroCult™ SM1 neuron supplement in 24-well or 96-well plates pre-coated with poly-D-lysine and laminin. Cells were maintained at 37°C and 5% CO2. Approximately 50 μL of conditioned medium was collected at different time points. The collected medium was stored at -20°C until the glutamate concentration was measured. The glutamate concentration was measured using 50 μL of conditioned medium for each sample. In time-course experiments, the glutamate concentration in the conditioned medium was measured over a five-day culture period. After 30 minutes of incubation, absorbance was measured on a SpectraMax® iD3 multimode microplate reader (from Molecular Devices, San Jose, CA).

[0102] Viability testing Approximately 50 μL of conditioned medium (CM) was used to determine glutamate concentration and cell viability. For this assay, the PrestoBlue™ High Sensitivity (HS) Cell Viability Reagent (from Thermo Fisher Scientific, Waltham, MA) was used, and the absorbance of each sample was read after 2 hours of incubation following the manufacturer’s manual procedure.

[0103] Inhibition of glutaminase Vandefitemcel (or SB623) cells were cultured in α-MEM supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2 for 16–24 hours. Next, the vandefitemcel (or SB623) cells were treated with two different concentrations of the glutaminase inhibitor Telaglenastat (CB-839; from Selleckchem, Houston TX): 5 μM and 50 μM, for two or three days. The vandefitemcel (or SB623) cells were washed twice and frozen for subsequent co-culture with glutamatergic neurons for MEA experiments. In parallel, 1.0 x 10⁻⁶ cells were cultured in α-MEM supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO₂ for 16–24 hours. 4 One vandefitemcel (or SB623) cell line was incubated with CB-839 at 37°C and 5% CO2 for 2 days. Cells were washed twice with PBS and cultured for three days in BrainPhys™ medium supplemented with NeuroCult™ SM1 neuronal complement at 37°C and 5% CO2. The level of glutamate in the conditioned medium was assessed.

[0104] Statistical analysis Statistical analyses were performed using GraphPad® Prism version 9.4.1 (from GraphPad Software, San Diego, California, USA). Spearman correlation coefficients were used to evaluate correlation analysis with 95% confidence intervals. The significance of differences between groups was determined by one-way ANOVA followed by Holm-Sidak post-hoc tests, paired t-tests, or Mann-Whitney tests (two-tailed), as shown in the illustration. Analysis of variance (ANOVA) was used, followed by Tukey or Sidak post-hoc tests, or a mixed-effects model with Geisser-Greenhouse correction and Tukey post-hoc tests for comparisons across multiple groups and time points. Unless otherwise specified, all tests were two-tailed.

[0105] Electrophysiological activity and viability of vandefitemcel and iFbN To determine whether vandefitemcel (or SB623 cells) exhibited neuron-like electrophysiological behavior, the electrophysiological activity of vandefitemcel (or SB623 cells) and iFbN was evaluated as single / separate cultures under serum-free neurophysiological conditions using the Axion Biosystem MEA system

[21] . Figures 1A-1C As shown, the cell density was 2.5 x 10⁻⁶ during the eight-week culture period. 4Vandefitemcel (or SB623 cells) cells did not exhibit any spontaneous neuronal-like electrophysiological activity, as evidenced by the number of spikes, effective electrodes, and network bursts. However, after approximately three weeks of culture, iFbN (cell density of 3.0 x 10⁻⁶ cells) showed significant neuronal activity. 4 5.0 x 10 cells 4 (each cell) exhibits spontaneous neuron-like electrophysiological activity, as evidenced by the number of spikes and the effectiveness of the electrodes (see [reference]). Figure 1A and Figure 1B iFbN also begins to exhibit spontaneous neuronal oscillations in the form of network bursts at approximately four to five weeks of cell culture (see [link]). Figure 1C When iFbN cells were plated at a higher cell density, they exhibited earlier and more pronounced neuronal oscillations compared to cells plated at a lower cell density, although the difference was not statistically significant (see [link to relevant documentation]). Figure 1E ).

[0106] Viability of vandefitemcel (or SB623 cells) and iFbN cells under neurophysiological conditions was evaluated using impedance modules available in the Axion Biosystem MEA system. Resistance (in ohms, Ω) between cells and electrodes was measured. In suspension, vandefitemcel (or SB623 cells) were large (approximately 17.2 μm in diameter) and 100% confluent, covering all 16 electrodes. In contrast, iFbN cells in suspension were smaller (approximately 10.2 μm in diameter) and formed clusters interconnected with cell processes when attached to MEA pores. These specific features distinguished vandefitemcel (or SB623 cells) and iFbN cells and resulted in significantly different resistances, as observed during the first week of culture (see [link to relevant documentation]). Figure 1D Although resistivity assessment using the MEA system indicated that vandefitemcel (or SB623 cells) survived in BrainPhys™ medium, resistivity was significantly reduced (32.6%) during the first three weeks of culture, and continued to decrease to 58.8% by week eight. On the other hand, iFbN cells were observed to exhibit increased resistivity (14% and 28.9%, respectively) in weeks two and three, compared to resistivity observed in the first week of culture, at both low and high cell densities (see [link to study]). Figure 1DIn the third week, and after the withdrawal of the maturation supplement, the resistivity in the iFbN cultures decreased slightly and then remained stable until week 5 of culture, at which point the resistivity began to increase again. This increased resistivity observed in the iFbN cell cultures indicates the presence of progenitor cells in these neuronal cultures. In fact, at week 5 of culture, 2.25% of the iFbN cell cultures were EdU-positive 24 hours after EdU labeling (see...). Figure 2C Despite the increased electrical resistance in iFbN cell cultures, there was no correlation between resistance and spike count at low or high iFbN cell seeding (Spearman correlation, r = 0.0665 and r = -0.1189, respectively), indicating that the increased neuronal activity cannot be explained by the presence of more cells within the MEA system.

[0107] Figure 1E It is 3.0 x 10 4 5.0 x 10 cells 4 A raster plot of iFbN cells at cell density, representing the electrophysiological activity of these cells as spikes and network bursts. Individual spikes are indicated by black boxes, electrode bursts by white boxes, and network bursts by dashed boxes.

[0108] In summary, vandefitemcel (or SB623 cells) survived in the MEA system but did not exhibit neuron-like electrophysiological activity, while iFbN cells exhibited some electrophysiological activity, as demonstrated by increased spike counts, effective electrodes, and bursts of neuronal networks in the Axion Biosystem MEA system.

[0109] Example The following examples are provided to illustrate various implementations of this disclosure. They are not intended to limit or restrict the entire scope of this disclosure. It should be understood that this disclosure is not limited to the specific embodiments described and illustrated herein, but includes all modifications and variations that fall within the scope of this disclosure as defined in the appended embodiments.

[0110] Example 1: Co-culturing vandefitemcel with iFbN cells led to increased neuronal activity and synapse formation in a cell density-dependent manner. Figures 2A-2E This shows that when co-cultured with iFbN cells, vandefitemcel (or SB623 cells) in... Figures 2A-2E (Shown as SB) promotes neuronal activity / excitability and synapse formation. Figure 2AImmunocytochemical (ICC) staining of vandefitemcel and iFbN cell co-cultures, where iFbN cells express the neuronal marker MAP2, vandefitemcel (or SB623 cells) express green fluorescent protein (GFP), and the cell nuclei are stained with 4',6-diamidindo-2-phenylindole (DAPI). iFbN cells were cultured at 5 x 10⁻⁶ cells / year. 4 Cells were plated at a density of 100 cells / year and, 14 days after maturation, were seeded at two cell densities (2.0 x 10⁻⁶ cells / year). 3 1 cell and 7.0 x 10 3 Add vandefitemcel to each cell. Figure 2A As shown, vandefitemcel cells (shown as SB-GFP) were detected in the culture of MAP2-positive forebrain neurons after 30 days of co-culture.

[0111] Figure 2B As shown, the presence of vandefitemcel (or SB623 cells, denoted as "SB" in the figure) contributes to the higher cell density (7.0 x 10⁻⁶). 3 This was confirmed by an increase in resistance measured between the cells and the MEA electrode, measured only one day after the start of co-culture in vandefitemcel (7.0 x 10⁻⁶ cells). 3 In the fourth week of co-culture of vandefitemcel (1 cell), an increase in electrical resistance was observed.

[0112] To evaluate whether vandefitemcel affects the neuronal electrical activity of iFbN, iFbN control cultures (in...) were used... Figure 2C The number of peaks in (displayed as "iFbN") was compared with the number of peaks in co-cultures of iFbN and vandefitemcel at different cell densities (in... Figure 2C In the middle, iFbN cells with 2.0 x 10 3 The co-culture of vandefitemcel cells was shown as "iFbN+SB (2k)", and the iFbN cells were cultured with 7.0 x 10⁻⁶ cells. 3 The coculture of vandefitemcel cells was shown as “iFbN+SB (7k)”. Figure 2C This indicates that, based on the observed difference in the number of spikes, an increase in neuronal excitability was detected in the co-culture of iFbN and vandefitemcel relative to the iFbN culture after two weeks of co-culture. Figure 2CAs shown, the number of spikes, or the degree of neuronal excitability, increased in a cell density-dependent manner. The difference in the number of spikes between the iFbN control culture and the iFbN+SB (7k) co-culture was statistically significant (one-way ANOVA Holm-Sidak). p<0.005).

[0113] To determine whether the increased neuronal activity promoted by vandefitemcel (or SB623 cells) also increased synapse formation, cultures of iFbN cells (in...) were also tested. Figure 2D The cells were displayed as “iFbN”, and the iFbN cells were 2.0 x 10⁻⁶. 3 A co-culture of vandefitemcel cells (shown as "iFbN+SB (2k)") and iFbN cells with 7.0 x 10⁻⁶ cells. 3 The number of network bursts was counted in a co-culture of vandefitemcel cells (shown as “iFbN+SB (7k)”). Figure 2D The results showed that adding vandefitemcel to iFbN at four weeks of co-culture also increased the number of network bursts in a cell density-dependent manner. During the next eight weeks of co-culture, spike and network burst counts continued to increase, remaining distinguishable from the iFbN-only control. The difference in network burst count between either the iFbN control culture or the iFbN+SB co-culture was statistically significant (one-way ANOVA Holm-Sidak). p<0.05; p<0.005).

[0114] Previously, vandefitemcel (or SB623 cells) was reported to promote neuropoietics in embryonic rat neural cultures [22, 23]. Figure 2E The culture compared to the iFbN control culture (in) was shown. Figure 2E Compared to cells labeled "iFbN" in the image, iFbN cells were 7.0 x 10⁻⁶. 3 A co-culture of vandefitemcel cells (in) Figure 2E The increase in glial fibrillary acidic protein (GFAP+) cells was shown in the data as “iFbN+SB(7k)”. Figure 2EFlow cytometry analysis showed that the GFPA+ population in the iFbN+SB(7k) co-culture was ~28.2%, compared to ~15.82% in the iFbN control culture. The EdU+ population was comparable between the iFbN+SB(7k) co-culture and the iFbN control culture. This indicates that vandefitemcel (or SB623 cells) promotes greater glial differentiation of forebrain neurons.

[0115] Example 2: Compared with co-culturing iFbN cells with astrocytes, co-culturing vandefitemcel with iFbN cells resulted in more neuronal activity, and molecules secreted by vandefitemcel increased neuronal activity. Cocultures of vandefitemcel (or SB623 cells) and iFbN were compared with cocultures of human astrocytes (hA) and iFbN to investigate whether the increased neuronal activity was induced by additional cells in the MEA system. Furthermore, iFbN were cultured in vandefitemcel conditioned medium (CM) to evaluate whether secreted factors had similar effects on neuronal activity. Conditioned medium was collected daily from vandefitemcel (or SB623 cells) cultured in BrainPhys™ medium and added to the iFbN cell culture immediately upon medium replacement. iFbN cells were cultured for two weeks, followed by the addition of other cell types; at two weeks, vandefitemcel, vandefitemcel conditioned medium, and human astrocytes were added, and spikes and network bursts were counted in each case.

[0116] like Figures 3A-3C As shown, in the two weeks following the addition of vandefitemcel to iFbN cells, the co-culture of vandefitemcel and iFbN (in...) Figures 3A-3C The cells shown as “iFbN+SB” are compared to iFbN cells alone (in the text). Figures 3A-3C (displayed as "iFbN") produces significantly more spikes (see...) Figure 3A One-way ANOVA Holm-Sidak p<0.01) and network bursts (see Figure 3B One-way ANOVA Holm-Sidak p<0.05, and Figure 3C In addition, in Figures 3A-3C As can be seen from this, the conditioned medium of vandefitemcel and the co-culture of iFbN (in) Figures 3A-3CThe co-culture of human astrocytes and iFbN (displayed as "iFbN+CM") also produced more spikes and network bursts than iFbN cells alone (although to varying degrees as iFbN+SB). Furthermore, co-cultures of human astrocytes and iFbN (in...) Figures 3A-3C The iFbN+hA cells also produced more spikes and network bursts than iFbN cells alone (although to a different degree than iFbN+SB).

[0117] Figure 3C This is a raster plot showing the electrophysiological activity of electrodes across the MEA system at four weeks of culture of individual iFbN cultures and co-cultures of iFbN with vandefitemcel, vandefitemcel conditioned medium (CM), and human astrocytes (hA). Individual spikes are indicated by black boxes, electrode bursts by white boxes, and network bursts by dashed boxes. In summary, these results indicate that molecules secreted from vandefitemcel lead to increased neuronal activity, but the presence of vandefitemcel cells in the culture significantly enhances this effect.

[0118] Example 3: Co-culturing vandefitemcel with glutamatergic neurons resulted in increased neuronal activity. Neuronal network bursts, or neuronal oscillations, are generated by intrinsic neuronal connections between excitatory and inhibitory neurons. Figure 4A Immunocytochemical (ICC) staining of iFbN cultures at 30 days of maturation revealed neurons expressing the neuronal marker MAP2, as well as the excitatory vesicular glutamate transporter 1 (vGLUT1) and the inhibitory γ-aminobutyric acid (GABA). Nuclei were stained with DAPI (scale: 50 µm). ICC staining showed abundant expression of vGLUT1, indicating the presence of excitatory neurons. This observation was confirmed by a decrease in peak activity upon addition of kynurenic acid (50 μM) (a broad-spectrum glutamate receptor antagonist) to the iFbN cultures.

[0119] To investigate the effects of vandefitemcel (or SB623) cells on a population of excitatory neurons, vandefitemcel was co-cultured with rich clusters of iPSC-derived glutamatergic neurons (hereinafter referred to as "glutamatergic neurons"). Unlike iFbN used in previous experiments, glutamatergic neurons mature on day 1 of culture and do not require a two-week maturation period. Therefore, glutamatergic neurons and vandefitemcel were co-cultured on day 1. Vandefitemcel has an effect on glutamatergic neurons (in... Figures 4B-4E The role of glutamatergic neurons (displayed as "GluN+SB") is similar to that of individual glutamatergic neurons (in...) Figures 4B-4D Glutamate neurons (displayed as "GluN") or co-cultured with human astrocytes at the same cell density as vandefitemcel (or SB623 cells) Figures 4B-4D The comparison is shown as “GluN+hA” in the middle.

[0120] Figure 4B The study showed that on day 3 of co-culture, vandefitemcel promoted high levels of spiking activity in glutamatergic neurons compared to spiking activity in individual glutamatergic neurons or in co-cultures of glutamatergic neurons and human astrocytes (unidirectional ANOVA Holm-Sidak). p < 0.0001). Figure 4C The study showed that this spike activity continued to increase for two weeks, while human astrocytes (hA) slightly promoted spike activity from the third week onwards in co-culture with glutamatergic neurons (bidirectional ANOVA Tukey). p<0.05; p<0.001; p<0.0001). Figure 4D The study showed that vandefitemcel (or SB623 cells) induced more network burst activity in the first week of co-culture with glutamatergic neurons (GluN+SB), while glutamatergic neurons co-cultured with human astrocytes (GluN+hA) did not show network burst activity until the fourth week (bidirectional ANOVA Tukey). p<0.05; p;<0.005; p<0.001; p<0.0001). Figure 4D and Figure 4E The study also showed that network bursts in a co-culture of glutamatergic neurons and vandefitemcel (GluN+SB) decreased after the first week, indicating the presence of inhibitory inputs in the culture. Figure 4C and Figure 4DThe study showed that the culture of glutamatergic neurons alone (GluN) had lower spike activity and failed to form a network burst after five weeks of culture compared to a co-culture of human astrocytes (GluN+hA) or vandefitemcel (GluN+SB).

[0121] Figure 4E This is a raster plot showing the electrophysiological activity of the GluN+SB co-culture and the progressive network burst development of electrodes across the MEA system. Numerous spikes were observed in the GluN+SB co-culture on day 3 (D3). Numerous spikes and network bursts were observed in the GluN+SB co-culture on day 6 (D6). On day 27 (D27), the spike count and network burst count decreased, indicating inhibitory synaptic input between network bursts. Individual spikes are indicated by black boxes in the plot, electrode bursts by white boxes, and network bursts by dashed boxes.

[0122] To investigate the effects of vandefitemcel (or SB623) cells on GABAergic neurons, the same experiment was repeated using rich clusters of iPSC-derived GABAergic neurons (hereinafter, "GABAergic neurons"). Figure 4F and Figure 4G As shown, when compared to individual GABAergic neurons (shown as "GABA-N") or a co-culture of GABAergic neurons and human astrocytes (shown as "GABA-N+hA"), vandefitemcel (or SB623 cells) promoted spikes and network bursts of GABAergic neurons at weeks 2 and 4 of co-culture (bidirectional ANOVA Tukey). p < 0.05; p < 0.005; (p < 0.0001). Although the number of network bursts induced by vandefitemcel in co-culture with GABAergic neurons reached levels similar to those observed in co-culture with glutamatergic neurons, it took longer to reach that peak (see [link]). Figure 4E and Figure 4H Similar to glutamatergic neurons, isolated GABAergic neurons did not exhibit network bursts, suggesting a need for supporting cells to promote electrical activity in GABAergic neurons. Interestingly, a decrease in electrical resistance was observed during the first week of culture, followed by recovery in the second week, and then a sustained decline until the fifth week. This pattern mirrors observations in individually cultured vandefitemcel (or SB623 cells) (see [link to relevant documentation]). Figure 1DThese findings suggest that vandefitemcel (or SB623 cells) induces earlier and higher spike activity in enriched glutamatergic neurons, indicating that SB623 cells play a role in regulating synaptic activity in excitatory neurons.

[0123] Figure 4H This is a raster plot showing the electrophysiological activity of the GABA-N+SB co-culture and the progressive network burst development of electrodes across the MEA system. No spike activity was observed on day 3 of the GABA-N+SB623 co-culture (not shown). Numerous spikes were observed in the GABA-N+SB co-culture on day 6 (D6). Numerous spikes and network bursts were observed in the GABA-N+SB co-culture on day 27 (D27). Individual spikes are indicated by black boxes in the plot, electrode bursts by white boxes, and network bursts by dashed boxes.

[0124] Example 4: vandefitemcel increases neuronal activity and promotes synaptic plasticity through tonic glutamate release. Glutamate is a major excitatory neurotransmitter in the mammalian nervous system and is involved in synapse formation

[24] . We first observed the amount of glutamate released by vandefitemcel by comparing the level of glutamate in the conditioned medium (CM) of vandefitemcel (or SB623 cells) with the level of glutamate in the CM of human astrocytes (hA). Vandefitemcel (or SB623 cells) and human astrocytes were cultured in BrainPhys™ medium at different cell densities for 5 days.

[0125] Figure 5A The results showed that at two days of culture, the increased CM glutamate levels in both cell types were cell density-dependent, but the increase was not observed in vandefitemcel (or SB623 cells) in the latter two cell types. Figure 5A The glutamate level in CM (displayed as "SB") is higher than that in human astrocytes (in... Figure 5A The glutamate level in CM (shown as "hA") is about 10 times higher (double ANOVA Holm-Sidak, p<0.001; p<0.0001).

[0126] Figure 5B This shows that at 5 days of culture, vandefitemcel (or SB623 cells, in) Figure 5BThe glutamate level in CMs (shown as "SB") increased 4.9-fold, while that in human astrocytes (in...) Figure 5B The glutamate level in CM (shown as "hA") increased 2.7-fold, indicating that the stress glutamate release from vandefitemcel was higher than that in human astrocytes (bidirectional ANOVA Holm-Sidak). p<0.05; p<0.005; p<0.001).

[0127] Glutamate is catalyzed by glutaminase in the release of glutamine. To investigate whether glutaminase plays a role in glutamate release from vandefitemcel (or SB623 cells), glutamine was added to BrainPhys™ medium and glutamate levels in the CM were assessed. After two days of culture, glutamate levels in the CM increased 1.6-fold, indicating that glutaminase does indeed play a role in the release of glutamate from vandefitemcel.

[0128] Next, experiments were conducted to determine whether inhibition of glutaminase in vandefitemcel (or SB623 cells) would affect the excitability observed in co-cultures of vandefitemcel and glutamatergic neurons (GluNs). To this end, glutaminase activity in vandefitemcel was inhibited using Telaglenastat (CB-839). Vandefitemcel (or SB623 cells) were incubated with CB-839 for two days and maintained in BrainPhys™ medium for three days to assess glutamate levels in the CM.

[0129] Figure 5C This is a comparison with the control group of vandefitemcel incubated with dimethyl sulfoxide (DMSO). Figure 5C Compared to 5 μM and 50 μM CB-839, the level of glutamate in vandefitemcel CM after incubation with CB-839 was shown as "SB (DMSO)". Figure 5C The results, displayed as "SB (CB-839-5 μM)" and "SB (CB-839-50 μM)", showed reductions of 1.5 times and 2.0 times respectively (unidirectional ANOVA Holm-Sidak). p < 0.0001).

[0130] Glutamate can participate in cellular metabolism, where it is converted to α-ketoglutarate and then enters the tricarboxylic acid cycle to produce ATP

[25] . Therefore, to evaluate whether inhibition of glutaminase would affect cellular metabolism and viability, the viability of vandefitemcel (or SB623 cells) treated with CB-839 was assessed using the PrestoBlue™ High Sensitivity (HS) Cell Viability Tester (from Thermo Fisher Scientific, Waltham, MA). Figure 5D This indicates that incubation with CB-839 does not alter the viability of vandefitemcel (or SB623 cells) (one-way ANOVA Holm-Sidak, “ns” = no difference in cell viability).

[0131] Next, glutamatergic neurons were co-cultured with vandefitemcel pre-incubated with 5 μM and 50 μM CB-839 to inhibit glutaminase. Figure 5E and Figure 5F The results showed that after one week of co-culture, the control culture of vandefitemcel incubated with dimethyl sulfoxide (DMSO) was significantly different from that of the control culture. Figure 5E and Figure 5F Compared to “SB (DMSO)”, inhibition of glutaminase in vandefitemcel significantly reduced spiking activity in glutamatergic neurons and co-cultures of vandefitemcel (shown as “GluN+SB (CB-839-5 μM)” and “GluN+SB (CB-839-50 μM)”) (see [link to article]). Figure 5E ) and network outbreaks (see Figure 5F (One-way ANOVA Holm-Sidak, p < 0.0005; p < 0.0001).

[0132] Finally, experiments were conducted to investigate whether human astrocytes (hA) could reduce the effect of vandefitemcel on glutamatergic neurons. Astrocytes play a key role in brain homeostasis by taking up glutamate from the extracellular space

[26] . Therefore, three co-cultures were prepared, including 1) glutamatergic neurons with vandefitemcel (in Figure 5G and Figure 5H (displayed as "GluN+SB"), 2) Glutamate neurons with vandefitemcel and human astrocytes (in Figure 5G and Figure 5H The text appears to be a mix of seemingly unrelated fragments and incomplete sentences, making it impossible to translate accurately. It seems to be discussing glutamatergic neurons and human astrocytes, possibly related to neuronal relationships and their interactions. Figure 5G and Figure 5H (Displayed as "GluN+hA"). Neuronal activity and synaptic plasticity are assessed in terms of the number of spikes (i.e., action potentials that appear in the signal when neurons fire) and the number of network bursts.

[0133] Figure 5G and Figure 5H It is said that star-shaped glial cells do indeed reduce the number of spikes and network bursts otherwise induced by vandefitemcel (or SB623 cells) on glutamatergic neurons (unidirectional ANOVA Holm-Sidak, (p<0.05). This data suggests that the increased neuronal activity and synaptic plasticity driven by vandefitemcel are induced by tonic glutamate release.

[0134] Figure 5I This is a schematic diagram showing that vandefitemcel (or SB623 cells) promotes increased neuronal activity and synaptic plasticity through tonic glutamate release.

[0135] The experimental results disclosed in this paper demonstrate that vandefitemcel (or SB623 cells), a genetically modified bone marrow-derived MSC, increases neuronal activity and synaptic plasticity in human neurons, and that this effect is induced by tonic glutamate release from vandefitemcel. As will be discussed in the following sections, the findings from these experiments can be applied to MSC-based cell therapies involving the transplantation of vandefitemcel (or SB623 cells) in human and animal subjects.

[0136] Furthermore, functional magnetic resonance imaging (fMRI)

[27] can be used prior to cell transplantation to help determine the optimal target brain region for transplantation. For example, fMRI can be used to select brain regions that show neuronal activity, and vandefitemcel can be applied to such regions of the brain selected based on fMRI scans. This can lead to better efficacy of the cell transplantation procedure and a reduction in clinical outcome variability and treatment-critical adverse events.

[0137] Treatment One unexpected result from the experiments described in this paper is that introducing vandefitemcel into neurons induces, causes, or triggers tonic release of glutamate into such neurons. Another unexpected result from the experiments described in this paper is that introducing vandefitemcel into neurons increases synapse formation in such neurons.

[0138] Based on the experimental results disclosed herein, a method for inducing, eliciting, or triggering tonic release of glutamate in subjects includes applying vandefitemcel to brain regions of subjects, wherein the vandefitemcel is derived from mesenchymal stem cells (MSCs) transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD).

[0139] Administration of vandefitemcel can also include administration via intrabrain implantation. When implanted via intrabrain implantation, vandefitemcel can release glutamate to neurons in the brain.

[0140] Administration of vandefitemcel may also include injection of vandefitemcel at multiple sites within the subject's brain. In some cases, vandefitemcel can be administered stereotactically through a burr hole in the subject's skull.

[0141] Further details regarding stereotactic application of cells can be found in U.S. Patent No. 11,439,761, the contents of which are incorporated herein by reference in their entirety for the purpose of describing stereotactic application of vandefitemcel and devices for such purposes.

[0142] The administration of vandefitemcel may also include applying vandefitemcel to a site in the forebrain of the subject, said site including at least one of the subject’s cerebrum, thalamus, and hypothalamus.

[0143] Administration of vandefitemcel may also include administering vandefitemcel to the subject's hippocampus.

[0144] The administration of vandefitemcel may also include applying vandefitemcel to the site of injury or disease in a region of the subject's brain.

[0145] Administration of vandefitemcel may include administration via parenteral administration.

[0146] Vandefitemcel can be suspended in a sterile isotonic crystalloid solution. For example, cell suspensions can contain cells suspended in Plasma-Lyte™ A (Baxter Healthcare Corporation). Cells can also be suspended in another physiologically compatible carrier, such as phosphate-buffered saline.

[0147] The effective therapeutic dose of vandefitemcel can be between approximately 1 million cells and 10 million cells (e.g., approximately 1 million cells, 1.5 million cells, 2 million cells, 2.5 million cells, 3 million cells, 3.5 million cells, 4 million cells, 4.5 million cells, 5 million cells, 5.5 million cells, 6 million cells, 6.5 million cells, 7 million cells, 7.5 million cells, 8 million cells, 8.5 million cells, 9 million cells, 9.5 million cells, 10 million cells, and amounts in between).

[0148] As disclosed herein, vandefitemcel can be prepared by a method comprising: (i) providing a culture of mesenchymal stem cells; (ii) contacting the culture of said mesenchymal stem cells with a polynucleotide encoding a Notch intracellular domain (NICD), wherein said polynucleotide does not encode a full-length Notch protein; (iii) selecting cells containing said polynucleotide; and (iv) further culturing the selected cells in the absence of selection for the polynucleotide. The mesenchymal stem cells may be human bone marrow-derived cells.

[0149] As disclosed herein, the method may further include capturing a functional magnetic resonance imaging (fMRI) scan of a brain region of a subject, selecting a brain region displaying neuronal activity based on the fMRI scan, and applying vandefitemcel to said brain region.

[0150] As disclosed herein, vandefitemcel can be prepared by a method comprising: (i) providing a culture of mesenchymal stem cells; (ii) contacting the culture of said mesenchymal stem cells with a polynucleotide encoding a Notch intracellular domain (NICD), wherein said polynucleotide does not encode a full-length Notch protein; (iii) selecting cells containing said polynucleotide; and (iv) further culturing the selected cells in the absence of selection for the polynucleotide. The mesenchymal stem cells may be human bone marrow-derived cells.

[0151] As disclosed herein, the method may further include capturing a functional magnetic resonance imaging (fMRI) scan of a brain region of a subject, selecting a brain region displaying neuronal activity based on the fMRI scan, and applying vandefitemcel to said brain region.

[0152] A method for increasing synapse formation in neurons is also disclosed, comprising applying vandefitemcel to a brain region of a subject containing neurons, wherein the vandefitemcel is derived from mesenchymal stem cells (MSCs) transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD).

[0153] Administration of vandefitemcel can also include administration via intrabrain implantation. When implanted via intrabrain implantation, vandefitemcel can release glutamate to neurons in the brain.

[0154] Administration of vandefitemcel also includes injections at multiple sites within the subject's brain. In some cases, vandefitemcel can be administered stereotactically through a borehole in the subject's skull.

[0155] Administration of vandefitemcel may also include administering vandefitemcel to the subject's hippocampus.

[0156] The administration of vandefitemcel may also include applying vandefitemcel to the site of injury or disease in a region of the subject's brain.

[0157] Administration of vandefitemcel may include administration via parenteral administration.

[0158] Vandefitemcel can be suspended in a sterile isotonic crystalloid solution. For example, cell suspensions can contain cells suspended in Plasma-Lyte™ A (Baxter Healthcare Corporation). Cells can also be suspended in another physiologically compatible carrier, such as phosphate-buffered saline.

[0159] The effective therapeutic dose of vandefitemcel can be between approximately 1 million cells and 10 million cells (e.g., approximately 1 million cells, 1.5 million cells, 2 million cells, 2.5 million cells, 3 million cells, 3.5 million cells, 4 million cells, 4.5 million cells, 5 million cells, 5.5 million cells, 6 million cells, 6.5 million cells, 7 million cells, 7.5 million cells, 8 million cells, 8.5 million cells, 9 million cells, 9.5 million cells, 10 million cells, and amounts in between).

[0160] As disclosed herein, vandefitemcel can be prepared by a method comprising: (i) providing a culture of mesenchymal stem cells; (ii) contacting the culture of said mesenchymal stem cells with a polynucleotide encoding a Notch intracellular domain (NICD), wherein said polynucleotide does not encode a full-length Notch protein; (iii) selecting cells containing said polynucleotide; and (iv) further culturing the selected cells in the absence of selection for the polynucleotide. The mesenchymal stem cells may be human bone marrow-derived cells.

[0161] As disclosed herein, the method may further include capturing a functional magnetic resonance imaging (fMRI) scan of a brain region of a subject, selecting a brain region displaying neuronal activity based on the fMRI scan, and applying vandefitemcel to said brain region.

[0162] A method for inducing, eliciting, or triggering glutamate release in subjects is also disclosed. This method may include capturing functional magnetic resonance imaging (fMRI) scans of brain regions of the subject, selecting brain regions displaying neuronal activity based on the fMRI scans, and administering vandefitemcel to the selected brain regions. Vandefitemcel is derived from mesenchymal stem cells (MSCs) transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD). Upon administration via intrabrain implantation, vandefitemcel releases glutamate.

[0163] A method for increasing synapse formation in neurons is also disclosed. This method may include capturing functional magnetic resonance imaging (fMRI) scans of brain regions of a subject, selecting brain regions displaying neuronal activity based on the fMRI scans, and administering vandefitemcel to the selected brain regions containing neurons. Vandefitemcel is derived from mesenchymal stem cells (MSCs) transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD). After administration via intrabrain implantation, vandefitemcel releases glutamate into neurons.

[0164] Compositions, formulations and kits The composition can increase synapse formation in neurons and may contain vandefitemcel in amounts between approximately 1 million and 10 million cells. Vandefitemcel can be produced by modifying mesenchymal stem cells derived from bone marrow. The composition may also contain one or more pharmaceutically acceptable excipients.

[0165] As previously discussed, vandefitemcel can be derived from mesenchymal stem cells transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD). Vandefitemcel can be prepared by a process involving providing a culture of mesenchymal stem cells (i.e., human bone marrow-derived cells) and contacting the culture of mesenchymal stem cells with a polynucleotide encoding the NICD. In some cases, the polynucleotide does not encode the full-length Notch protein. Vandefitemcel can be further prepared by selecting cells containing said polynucleotide; and, in the absence of selection for said polynucleotide, further culturing the selected cells.

[0166] The composition may also contain additional mesenchymal stem cells that have not been transiently transfected with polynucleotides.

[0167] Mesenchymal stem cells can be transiently transfected using plasmid vectors containing polynucleotides encoding NICD.

[0168] Vandefitemcel can be suspended in a sterile isotonic crystalloid solution.

[0169] One or more pharmaceutically acceptable excipients may include at least one of buffers, proteins, stabilizers, and preservatives. One or more pharmaceutically acceptable excipients may also contain a carrier or diluent to form a cell suspension.

[0170] Pharmaceutically acceptable carriers can be physiologically compatible carriers intended for implantation. As used herein, the term "physiologically compatible carrier" can refer to a carrier that is compatible with other components of the formulation and is harmless to its recipient. Examples of suitable carriers or diluents include cell culture media (e.g., Eagle Minimum Essential Medium), phosphate-buffered saline, Hank balanced salt solution + / - glucose (HBSS), and various electrolyte solutions. Pharmaceutically acceptable carriers or diluents can also be or contain sterile isotonic crystalloid solutions, such as Plasma-Lyte™ A (Baxter Healthcare Corporation).

[0171] The composition may contain vandefitemcel packaged in a sealed vial. In some cases, the sealed vial may contain 0.3 mL of cell suspension with a cell concentration of approximately 8.5%. 10 6 Cells / mL. Optionally, the sealed vial may contain 0.3 mL of cell suspension with a cell concentration of approximately 17.0%. 10 6 Cells / mL.

[0172] Other examples of materials that can be used as pharmaceutically acceptable carriers or excipients are also disclosed, including: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic and compatible substances. Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, releasing agents, coating agents, sweeteners, flavoring agents and perfuming agents, preservatives and antioxidants may also be present in the composition.

[0173] Other examples of pharmaceutically acceptable transporters or excipients include substances that stimulate angiogenesis (“angiogenic agents”). In some cases, angiogenic agents can be proteins (e.g., fibroblast growth factor, platelet-derived growth factor, transforming growth factor α, hepatocyte growth factor, vascular endothelial growth factor, Hedgehog factor, MAGP-2, HIF-1, PR-39, RTEF-1, c-Myc, TFII, Egr-1, ETS-1) or nucleic acids encoding such proteins. See, for example, Vincent et al. (2007) Gene Therapy 14:781-789. In other cases, angiogenic agents can be small RNA molecules (e.g., siRNA, shRNA, microRNA) or ribozymes that target nucleic acids encoding angiogenesis inhibitors. Furthermore, angiogenic agents can be triple-stranded nucleic acids that bind to DNA sequences that regulate the expression of proteins that inhibit angiogenesis, thereby blocking the transcription of the gene encoding that protein.

[0174] Exemplary formulations include, but are not limited to, those suitable for parenteral administration, such as intrapulmonary, intra-arterial, intraocular, intracranial, subdural, or subcutaneous administration, including formulations encapsulated in micelles, liposomes, or drug-release capsules (with the active agent incorporated into a biocompatible coating designed for slow release); ingestible formulations; formulations for topical application, such as eye drops, creams, ointments, and gels; and other formulations, such as inhalers, aerosols, and sprays. The dosage of the compositions disclosed herein may vary depending on the degree and severity of therapeutic need, the activity of the applied composition, the general health condition of the subject, and other considerations well known to those skilled in the art.

[0175] In another embodiment, the compositions described herein may also be delivered locally. Local delivery allows for non-systemic delivery of the composition, thereby reducing the physical burden on the composition compared to systemic delivery. Such local delivery can be achieved, for example, by using various medical implantation devices, including but not limited to stents and catheters, or by inhalation, injection, or surgery. Methods for applying, implanting, embedding, and otherwise attaching the desired agent to medical devices such as stents and catheters are known in the art and are contemplated herein.

[0176] Another aspect of this disclosure relates to a kit for administering cells to a subject. For example, the kit may contain a suitably formulated (e.g., in a drug delivery vehicle) cell composition, which is present as one or more separate pharmaceutical products.

[0177] Compositions containing vandefitemcel can be used in combination with other compositions containing a substance that stimulates angiogenesis (“angiogenic agent”). The compositions can be administered sequentially or simultaneously in any order. Therefore, therapeutic compositions as disclosed herein can contain both vandefitemcel and an angiogenic agent. In another embodiment, a single therapeutic composition, one containing vandefitemcel and the other containing an angiogenic agent, can be administered to a subject alone or together.

[0178] Angiogenic agents can be transcription factors that activate the expression of pro-angiogenic molecules (e.g., proteins). Naturally occurring transcription factors that regulate the expression of pro-angiogenic proteins (such as, for example, HIF-1α) are known. Furthermore, synthetic transcriptional regulatory proteins can be constructed through genetic engineering. For example, methods for designing zinc finger DNA-binding domains that bind to sequences of interest, and methods for fusing such zinc finger DNA-binding domains with transcriptional activation and repression domains, have been described. See, for example, U.S. Patents 6,534,261; 6,607,882; 6,785,613; 6,794,136; 6,824,978; 6,979,539; 7,013,219; 7,177,766; 7,220,719; and 7,788,044. These methods can be used to synthesize non-naturally occurring proteins that activate the transcription of any gene encoding a pro-angiogenic protein. Furthermore, zinc finger transcriptional activators of the vascular endothelial growth factor (VEGF) gene have been described. See, for example, U.S. Patents 7,026,462; 7,067,317; 7,560,440; 7,605,140; and 8,071,564. Therefore, non-naturally occurring (i.e., synthetic) zinc finger proteins that activate VEGF gene transcription can be used in combination with vandefitemcel to enhance angiogenesis, for example, in the treatment of stroke. Additionally, natural or synthetic transcriptional regulatory proteins that inhibit the transcription of anti-angiogenic molecules (e.g., synthetic zinc finger transcriptional regulatory proteins) can also be used as pro-angiogenic agents.

[0179] Numerous embodiments have been described. However, those skilled in the art will understand that various changes and modifications can be made to this disclosure without departing from the spirit and scope of the embodiments. Elements of systems, apparatuses, devices, and methods shown in any embodiment are exemplary for a particular embodiment and can be used in combination or otherwise applied to other embodiments of this disclosure. For example, the steps of any method depicted in the drawings or described in this disclosure do not require a specific order or sequence shown or described to achieve the desired result. Furthermore, other steps or operations may be provided, or steps or operations may be eliminated or omitted from the described method or process to achieve the desired result. Additionally, any component or portion of any device or system described in this disclosure or depicted in the drawings may be removed, eliminated, or omitted to achieve the desired result. Furthermore, for the sake of brevity and clarity, certain components or portions of the systems, apparatuses, or devices shown or described herein have been omitted.

[0180] Therefore, other embodiments are within the scope of the appended claims, and the description and / or drawings may be regarded as illustrative rather than restrictive.

[0181] Each of the individual variations or embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any other variation or embodiment. Modifications can be made to adapt particular circumstances, materials, composition, processes, process actions, or steps to the purpose, spirit, or scope of the invention.

[0182] The methods described herein can be performed in any logically possible order of the described events, and in any order of events described. Furthermore, additional steps or operations may be provided, or steps or operations may be eliminated to achieve the desired result.

[0183] Furthermore, where a range of values ​​is provided, every intermediate value between the upper and lower limits of that range, as well as the value or intermediate value of any other statement within that range, is covered within the scope of this invention. Additionally, any optional features of the variations of the invention described herein may be set forth and claimed independently or in combination with any one or more of the features described herein. For example, a description of a range from 1 to 5 should be considered as having disclosed subranges such as from 1 to 3, from 1 to 4, from 2 to 4, from 2 to 5, from 3 to 5, etc., as well as individual numbers within that range such as 1.5, 2.5, etc., and any full or partial increments between them.

[0184] All existing subjects mentioned herein (e.g., publications, patents, patent applications) are incorporated herein by reference in their entirety, unless, to the extent currently possible, such subject matter might conflict with the subject matter of this invention (in which case the subject matter presented herein shall be accepted). Referenced items are provided solely because they were disclosed prior to the filing date of this application. Nothing herein should be construed as an admission that the invention is not qualified prior to such material due to prior invention.

[0185] References to singular items include the possibility of the existence of multiple identical items. More specifically, as used herein and in the appended claims, the singular forms “a,” “an,” “said,” and “the” include plural indicators unless clearly specified in the context. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a prior basis for the use of such exclusive terms as “solely,” “only,” etc., or the use of “negative” limitations in conjunction with references to claim elements. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0186] When the phrase “at least one of…” modifies more than one item or component (or a list of items or components), reference to such a phrase means any combination of one or more of these items or components. For example, the phrase “at least one of A, B, and C” means: (i) A; (ii) B; (iii) C; (iv) A, B, and C; (v) A and B; (vi) B and C; or (vii) A and C.

[0187] In understanding the scope of this disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open-ended terms that define the presence of stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers, and / or steps. The same applies to words with similar meanings, such as the terms "comprising," "having," and their derivatives. Furthermore, the terms "part," "section," "portion," "component," "element," or "part" when used in the singular can have a dual meaning of a single part or more than one part. As used herein, the following directional terms "forward, backward, above, downward, vertical, horizontal, below, laterally, and vertically," and any other similar directional terms, refer to those positions of parts of a device or apparatus or those directions in which parts of a device or apparatus are translated or moved.

[0188] Finally, degree terms such as “substantially,” “about,” and “approximately,” as used herein, refer to a specified value, or a specified value and a reasonable amount of deviation from the specified value (e.g., a deviation of ±0.1%, ±1%, ±5%, or ±10%, as such variation is appropriate), such that the final result will not be significantly or substantially changed. For example, “about 1.0 cm” can be interpreted as meaning “1.0 cm” or “between 0.9 cm and 1.1 cm.” When degree terms such as “about” or “approximately” are used to refer to numbers or values ​​that are part of a range, the term can be used to modify the minimum and maximum numbers or values.

[0189] This disclosure is not intended to be limited to the specific forms set forth herein, but rather to cover alternatives, modifications, and equivalents of the variations or embodiments described herein. Furthermore, the scope of this disclosure fully encompasses other variations or embodiments that may become apparent to those skilled in the art in light of this disclosure.

[0190] References 1. He, JQ, Sussman, ES, Steinberg, GK, 2020. Revisiting StemCell-Based Clinical Trials for Ischemic Stroke. Front Aging Neurosci 12,575990. 2. Volkman, R., Offen, D., 2017. Concise Review: Mesenchymal StemCells in Neurodegenerative Diseases. Stem Cells 35, 1867-1880. 3. Berebichez-Fridman, R., Montero-Olvera, PR, 2018. Sources and Clinical Applications of Mesenchymal Stem Cells: State-of-the-art review. Sultan Qaboos Univ Med J 18, e264-e277. 4. Kawabori, M., Weintraub, A.H., Imai, H., Zinkevych, L.,McAllister, P., Steinberg, G.K., Frishberg, B.M., Yasuhara, T., Chen, J.W.,Cramer, S.C., Achrol, A.S., Schwartz, N.E., Suenaga, J., Lu, D.C., Semeniv,I., Nakamura, H., Kondziolka, D., Chida, D., Kaneko, T., Karasawa, Y.,Paadre, S., Nejadnik, B., Bates, D., Stonehouse, A.H., Richardson, R.M.,Okonkwo, D.O., 2021. Cell Therapy for Chronic TBI: Interim Analysis of theRandomized Controlled STEMTRA Trial. Neurology 96, e1202-1214. 5. Steinberg, G.K., Kondziolka, D., Wechsler, L.R., Lunsford, L.D.,Kim, A.S., Johnson, J.N., Bates, D., Poggio, G., Case, C., McGrogan, M. andYankee, E.W., 2018. Two-year safety and clinical outcomes in chronic ischemicstroke patients after implantation of modified bone marrow-derivedmesenchymal stem cells (SB623): a phase 1 / 2a study. Journal of Neurosurgery,131(5), pp.1462-1472. 6. Wang, S., Hesen, R., Mossink, B., Kasri, N.N. and Schubert, D.,2023. Generation of glutamatergic / GABAergic neuronal co-cultures derived fromhuman induced pluripotent stem cells for characterizing E / I balance in vitro.STAR protocols, 4(1), p.101967. 7. De Filippi, G., Hayes, H.B., Nicolini, A.M., Arrowood, C.A. andMillard, D.C., 2020. Multiwell Microelectrode Array (MEA) Technology for thequantification of neuronal, synaptic, and network function for in vitroprimary and stem cell-derived neuronal models. Journal of Pharmacological andToxicological Methods, 105, p.106769. 8. Jiang, Yuehua, et al. Pluripotency of mesenchymal stem cellsderived from adult marrow. Nature 418.6893 (2002): 41-49. 9. Pittenger, Mark F., et al. Multilineage potential of adult humanmesenchymal stem cells. Science 284.5411 (1999): 143-147. 10. Dezawa, Mari, et al. Sciatic nerve regeneration in rats inducedby transplantation of in vitro differentiated bone-marrow stromal cells. European Journal of Neuroscience 14.11 (2001): 1771-1776. 11. Campagnoli, Cesare, et al. Identification of mesenchymal stem / progenitor cells in human first-trimester fetal blood, liver, and bonemarrow. Blood, The Journal of the American Society of Hematology 98.8 (2001):2396-2402. 12. Erices, Alejandro, Paulette Conget, and José J. Minguell.Mesenchymal progenitor cells in human umbilical cord blood. British journal of haematology 109.1 (2000): 235-242. 13. Artavanis-Tsakonas, Spyros, Kenji Matsuno, and Mark E. Fortini.Notch signaling. Science 268.5208 (1995): 225-232. 14. Mumm, Jeffrey S., and Raphael Kopan. Notch signaling: from theoutside in. Developmental biology 228.2 (2000): 151-165. 15. Ehebauer, Matthias, Penelope Hayward, and Alfonso Martinez-Arias.Notch signaling pathway. Science's STKE 2006.364 (2006): cm7-cm7. 16. Weinmaster, Gerry, Veronica J. Roberts, and Greg Lemke. A homologof Drosophila Notch expressed during mammalian development. Development 113.1(1991): 199-205. 17. Schroeter, Eric H., Jeffrey A. Kisslinger, and Raphael Kopan.Notch-1 signalling requires ligand-induced proteolytic release ofintracellular domain. Nature 393.6683 (1998): 382-386. 18. Dezawa, Mari, et al. Specific induction of neuronal cells frombone marrow stromal cells and application for autologous transplantation. The Journal of clinical investigation 113.12 (2004): 1701-1710. 19. Sambrook, Joseph, Edward F. Fritsch, and Tom Maniatis. Molecularcloning: a laboratory manual. No. Ed. 2. Cold spring harbor laboratory press,1989. 20. Brent, R., et al. Current Protocols in Molecular Biology(Janssen, K., ed) Vol. 1. (1994). 21. Bardy, C., van den Hurk, M., Eames, T., Marchand, C., Hernandez,R.V., Kellogg, M., Gorris, M., Galet, B., Palomares, V., Brown, J., Bang,A.G., Mertens, J., Bohnke, L., Boyer, L., Simon, S., Gage, F.H., 2015.Neuronal medium that supports basic synaptic functions and activity of humanneurons in vitro. Proc Natl Acad Sci U S A 112, E2725-2734. 22. Aizman, I., Tate, C.C., McGrogan, M., Case, C.C., 2009.Extracellular matrix produced by bone marrow stromal cells and by theirderivative, SB623 cells, supports neural cell growth. J Neurosci Res 87,3198-3206. 23. Aizman, I., Tirumalashetty, B.J., McGrogan, M., Case, C.C., 2014.Comparison of the neuropoietic activity of gene-modified versus parentalmesenchymal stromal cells and the identification of soluble and extracellularmatrix-related neuropoietic mediators. Stem Cell Res Ther 5, 29. 24. Mattson, M.P., 2008. Glutamate and neurotrophic factors inneuronal plasticity and disease. Ann N Y Acad Sci 1144, 97-112. 25. Hamanaka, R. B., O’Leary, E. M., Witt, L. J., Tian, Y., Gökalp,G. A., Meliton, A. Y., ... & Mutlu, G. M., 2019. Glutamine metabolism isrequired for collagen protein synthesis in lung fibroblasts. American journalof respiratory cell and molecular biology, 61(5), 597-606. 26. Romanos, J., Benke, D., Saab, A.S., Zeilhofer, H.U., Santello,M., 2019. Differences in glutamate uptake between cortical regions impactneuronal NMDA receptor activation. Commun Biol 2, 127. 27. Heeger, D. J., & Ress, D., 2002. What does fMRI tell us aboutneuronal activity?. Nature reviews neuroscience, 3(2), 142-151。

Claims

1. A method for inducing tonic release of glutamate in a subject, the method comprising: The vandefitemcel was administered to a brain region of the subject, wherein the vandefitemcel was derived from mesenchymal stem cells (MSCs) transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD).

2. The method of claim 1, wherein administering the vandefitemcel further comprises administering the vandefitemcel via intracerebral implantation.

3. The method of claim 2, wherein when implanted via intracerebral implantation, the vandefitemcel releases glutamate to neurons in the brain in a tonic manner.

4. The method of claim 2, wherein administering the vandefitemcel further comprises injecting the vandefitemcel at multiple sites within the brain region.

5. The method of claim 2, wherein administering the vandefitemcel further comprises stereotactic administration of the vandefitemcel via a borehole in the skull of the subject.

6. The method of claim 1, wherein the brain region is the forebrain of the subject.

7. The method of claim 1, wherein the brain region is the site of injury or disease.

8. The method of claim 1, wherein the brain region is the hippocampus of the subject.

9. The method of claim 1, wherein administering the vandefitemcel further comprises administering the vandefitemcel via parenteral administration.

10. The method of claim 1, wherein the vandefitemcel is suspended in a sterile isotonic crystalloid solution.

11. The method of claim 1, wherein applying the vandefitemcel further comprises applying between approximately 1 million and 10 million cells.

12. The method of claim 1, wherein the vandefitemcel is prepared by a method comprising: Provide MSC cultures; The MSC culture is contacted with the polynucleotide encoding NICD, wherein the polynucleotide does not encode the full-length Notch protein. Select cells containing the polynucleotide; and The selected cells are further cultured in the absence of selection for the polynucleotide.

13. The method of claim 1, wherein the MSC is a human bone marrow-derived cell.

14. The method according to claim 1, further comprising: Functional magnetic resonance imaging (fMRI) scans of brain regions captured by the subject; Based on the fMRI scan, a brain region was selected to display neuronal activity; and The vandefitemcel is applied to the brain region.

15. A method for increasing synapse formation in neurons, the method comprising: Vandefitemcel was administered to the brain region of the subject containing the neurons, wherein the vandefitemcel was derived from mesenchymal stem cells (MSCs) transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD).

16. The method of claim 15, wherein administering the vandefitemcel further comprises administering the vandefitemcel via intracerebral implantation.

17. The method of claim 16, wherein when implanted via intracerebral implantation, the vandefitemcel releases glutamate to the neuron in a tonic manner.

18. The method of claim 16, wherein administering the vandefitemcel further comprises injecting the vandefitemcel at multiple sites within the brain region.

19. The method of claim 16, wherein administering the vandefitemcel further comprises stereotactically administering the vandefitemcel via a borehole in the skull of the subject.

20. The method of claim 15, wherein the brain region is the forebrain of the subject.

21. The method of claim 15, wherein the brain region is a site of injury or disease.

22. The method of claim 16, wherein the brain region is the hippocampus of the subject.

23. The method of claim 15, wherein administering the vandefitemcel further comprises administering the vandefitemcel via parenteral administration.

24. The method of claim 15, wherein the vandefitemcel is suspended in a sterile isotonic crystal solution.

25. The method of claim 15, wherein applying the vandefitemcel further comprises applying between approximately 1 million and 10 million cells.

26. The method of claim 15, wherein the vandefitemcel is prepared by a method comprising: Provides cultures of mesenchymal stem cells; The mesenchymal stem cell culture is contacted with the polynucleotide encoding NICD, wherein the polynucleotide does not encode the full-length Notch protein. Select cells containing the polynucleotide; and The selected cells are further cultured in the absence of selection for the polynucleotide.

27. The method of claim 15, wherein the MSC is a human bone marrow-derived cell.

28. The method of claim 15, further comprising: Functional magnetic resonance imaging (fMRI) scans of brain regions captured by the subject; Based on the fMRI scan, a brain region was selected to display neuronal activity; and The vandefitemcel is applied to the brain region.

29. A composition for increasing synapse formation in neurons, the composition comprising: A quantity of vandefitemcel between approximately 1 million and 10 million cells, wherein the vandefitemcel is produced by modifying mesenchymal stem cells derived from bone marrow; and One or more pharmaceutically acceptable excipients.

30. The composition of claim 29, wherein the vandefitemcel is prepared by a process comprising: Provides cultures of mesenchymal stem cells; The mesenchymal stem cell culture is contacted with a polynucleotide encoding the Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full-length Notch protein. Select cells containing the polynucleotide; and The selected cells are further cultured in the absence of selection for the polynucleotide.

31. The composition of claim 30, wherein the mesenchymal stem cells are human bone marrow-derived cells.

32. The composition of claim 29, wherein the mesenchymal stem cells are transiently transfected with a plasmid vector containing a polynucleotide encoding NICD.

33. The composition of claim 29, wherein the one or more pharmaceutically acceptable excipients include at least one of buffers, proteins, stabilizers and preservatives.

34. The composition of claim 29, wherein the vandefitemcel is suspended in a sterile isotonic crystal solution.

35. The composition of claim 29, wherein the one or more pharmaceutically acceptable excipients comprise a carrier or a diluent.

36. A method for inducing glutamate release in a subject, the method comprising: Functional magnetic resonance imaging (fMRI) scans of brain regions captured by the subject; Based on the fMRI scan, brain regions displaying neuronal activity were selected; and Vandefitemcel is applied to a selected brain region, wherein the vandefitemcel is derived from mesenchymal stem cells (MSCs) transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD), and wherein the vandefitemcel releases glutamate upon administration via intrabrain implantation.

37. A method for increasing synapse formation in neurons, the method comprising: Functional magnetic resonance imaging (fMRI) scans of brain regions captured in the subjects; Based on the fMRI scan, brain regions displaying neuronal activity were selected; and Vandefitemcel is applied to selected brain regions containing the neurons, wherein the vandefitemcel is derived from mesenchymal stem cells (MSCs) transiently transfected with a polynucleotide encoding the Notch intracellular domain (NICD), and wherein the vandefitemcel releases glutamate into the neurons after administration via intrabrain implantation.

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