Methods and compositions for treating spinal cord injury

Abstract

Disclosed is a composition comprising a plurality of fibrous particles made from decellularized omentum, the fibrous particles having a diameter between 750 microns and 3 mm, the fibrous particles comprising a network of mature neurons. Also disclosed are uses thereof and methods of making thereof.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Application No. 63 / 283,629, filed November 29, 2021, the contents of which are incorporated by reference in their entirety herein.

[0002] Technical Field The present invention, in some embodiments thereof, relates to compositions for treating spinal cord injury, and more particularly, chronic spinal cord injury. [Background technology]

[0003] Traumatic spinal cord injury (SCI) has an immediate and devastating impact on motor control and all aspects of a patient's health and quality of life. The initial trauma causes direct damage, often leading to cell death, disruption of the blood-spinal cord barrier, and degradation of the extracellular matrix (ECM). These processes initiate a secondary inflammatory injury cascade, causing progressive chronic tissue damage and resulting in the formation of a glial scar. Although healthy neural tissue surrounding the injury site contains cues that can promote tissue repair, scar tissue and injury sites lack a permissive microenvironment for cell proliferation, as well as the absence of axon guidance molecules secreted by the ECM, such as netrin and slit, resulting in poor intrinsic regenerative capacity and permanent neurological dysfunction. Moreover, over time, the injury site expands, making natural or mediated regeneration even more challenging.

[0004] In an effort to rewire the injured spinal cord, researchers have transplanted various cell types or biomaterials into the site of acute injury. 5~7 Schwann cells have been proposed as a potential treatment for spinal cord injury. 8 , neural stem cells (NSCs) or neural progenitor cells (NPCs) 9、10 and mesenchymal stem cells 11However, there are two issues that could compromise the success of such therapies: host immune responses to allogeneic or xenogeneic cells that could promote cell rejection, and transplantation of dissociated cells that do not organize into functional networks.

[0005] To overcome the risk of rejection, induced pluripotent stem cells (iPSCs) may be used. In this approach, somatic cells from the patient are reprogrammed to become pluripotent and then differentiated into the desired cell lineage. The most common strategy in the regeneration of injured spinal cord is to transplant various iPSC-derived cell lines rather than directly applying these cells. Lu et al. inserted dissociated iPSC-derived NSCs into a fibrin matrix 2 weeks after SCI induction. The cells were able to differentiate and interact with host neurons to form axons that extended long distances through the white matter of the injured spinal cord. 13 In another study, iPSC-derived neurospheres were injected into the spinal cord 9 days after SCI. The cells differentiated in vivo into three neural lineages without forming teratomas and participated in remyelination and improved locomotion. 14 Although such a cell source may be relevant for the regeneration of injured spinal cord, injection of cells into the injury site is less than ideal. Injection of dissociated cells in suspension or in a biomaterial-based carrier into the injury site provides the energy to form cell-cell and cell-matrix interactions for tissue formation and differentiation, and for integration with healthy parts of the spinal cord. 15 Scar tissue does not provide a microenvironment that supports tissue construction, which can lead to massive cell death. Therefore, by inserting a preformed 3D neural network into the injury site after total or partial removal of the scar tissue, it is possible to shorten the time required for regeneration and improve the therapeutic effect. However, the conditions for engineering a functional 3D neural network are still not fully understood. 16 .

[0006] Background art includes Edri et al., Advanced materials 31, 1803895 (2019), Shimojo, et al. Mol Brain 8, 79 (2015), WO 2014 / 207744 and WO 2017 / 103930. Summary of the Invention

[0007] According to an aspect of the present invention, there is provided a composition comprising a plurality of fibrous particles made from decellularized omentum, the fibrous particles having a diameter between 750 microns and 3 mm, the fibrous particles comprising a network of mature neurons.

[0008] According to an aspect of the present invention, there is provided a method of treating a chronic spinal cord injury in a subject, comprising implanting a composition described herein at the site of injury in the subject at least three months after spinal cord injury, thereby treating the spinal cord injury.

[0009] According to an aspect of the present invention, (i) a composition as described herein; (ii) a device for delivering the composition to the spinal cord of a subject; and An article is provided that includes:

[0010] According to an aspect of the present invention, there is provided a method of making the composition described herein, comprising the steps of: (a) producing decellularized omentum particles containing pluripotent stem cells, the particles having a diameter of 750 microns to 3 mm; (b) contacting the particles with at least one neural cell differentiation agent; (b) culturing the particles in the presence of at least one neuronal differentiation agent under conditions that promote the generation of neural networks of mature neurons within the particles, thereby producing a composition as described herein; A method is provided, comprising:

[0011] According to an embodiment of the present invention, the fibrous particles are essentially spherical.

[0012] According to an embodiment of the present invention, the mature neurons include motor neurons.

[0013] According to an embodiment of the invention, greater than 50% of motor neurons express neuron-specific class III β-tubulin (TUJ1) as measured by flow cytometry.

[0014] According to an embodiment of the invention, greater than 50% of the cells express motor neuron and pancreas homeobox 1 (MNX1) as measured by flow cytometry.

[0015] According to an embodiment of the present invention, the average diameter of the fibers of the fibrous particles is 50 to 200 nm.

[0016] According to an embodiment of the present invention, the network includes a human network.

[0017] According to an embodiment of the invention, the composition further comprises a pharma- ceutically acceptable carrier.

[0018] According to an embodiment of the invention, the implantation is performed at least six months after the spinal cord injury.

[0019] According to an embodiment of the invention, the method further comprises removing scar tissue from the subject's injury site prior to implantation.

[0020] According to an embodiment of the invention, the composition comprises a pharma- ceutically acceptable carrier when implanted, and the method further comprises removing at least a portion of the carrier from the injury site after implantation.

[0021] According to an embodiment of the invention, implantation is performed using a syringe.

[0022] According to an embodiment of the present invention, the inner diameter of the syringe is 1 to 5 mm.

[0023] According to an embodiment of the invention, the device is a syringe.

[0024] According to an embodiment of the present invention, the inner diameter of the syringe is 1 to 5 mm.

[0025] According to an embodiment of the present invention, the pluripotent stem cells are induced pluripotent stem cells.

[0026] According to an embodiment of the present invention, the induced pluripotent stem cells are reprogrammed from omental stromal cells.

[0027] According to an embodiment of the present invention, the neural cell differentiation agent is selected from the group consisting of a transforming growth factor beta receptor 1 (ALK-5) inhibitor, a morphogenic protein 4 (BMP4) inhibitor, retinoic acid, bone derived neurotrophic factor (BDNF), ascorbic acid, and purmorphamine.

[0028] According to an embodiment of the present invention, the mature neurons include motor neurons.

[0029] According to an embodiment of the invention, greater than 50% of motor neurons express neuron-specific class III β-tubulin (TUJ1) as measured by flow cytometry.

[0030] According to an embodiment of the invention, greater than 50% of the cells express motor neuron and pancreatic homeobox 1 (MNX1) as measured by flow cytometry.

[0031] According to an embodiment of the present invention, the preparation comprises: (a) dispensing a droplet of a mixture of solubilized decellularized omentum and pluripotent stem cells onto a solid surface; (b) exposing the droplets to conditions that promote solidification of the droplets; This is carried out by.

[0032] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will prevail. Additionally, the materials, methods, and examples are illustrative only and are not necessarily intended to be limiting.

[0033] Certain embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings, in which: Reference will now be made specifically in detail to the drawings, it being stressed that the particulars shown are by way of example and are for the purposes of illustrative discussion of embodiments of the invention, and in which the description taken with reference to the drawings will make apparent to those skilled in the art how embodiments of the invention may be practiced. [Brief description of the drawings]

[0034] [Figure 1] 1A-B are schematic diagrams illustrating experiments performed according to an embodiment of the present invention. A. Concept. Omental tissue is removed from a patient. Cells and ECM are then separated. Cells are reprogrammed to become iPSCs and the ECM is processed into a thermoresponsive hydrogel. iPSCs are then encapsulated within an omentum-based hydrogel to create a stem cell implant. This implant undergoes a 30-day differentiation process that mimics embryonic spinal cord development. The resulting spinal neuron implant is fully autologous and can then be implanted back into the patient. B. Schematic of the study. Differentiated spinal motor neuron implants were first characterized in vitro. The therapeutic potential of these implants was then evaluated in hemisection acute and chronic SCI models. Molecular, behavioral and anatomical aspects were investigated. [Figure 2-1]Figure 2A-L are photographs showing the fabrication and analysis of spinal implants. A. Native omentum. B. Decellularized omentum. C. Omentum-based hydrogel. D. Rheological properties of omentum-based hydrogel. E. SEM imaging of acellular hydrogel. Scale bar = 2 μm. F. SEM imaging of iPSCs encapsulated within hydrogel. Scale bar = 5 μm. G. Flow cytometry analysis of undifferentiated iPSCs (TRA-1-60, SSEA-4) cultured within hydrogel for 3 days. H. Immunofluorescence imaging of undifferentiated iPSCs cultured within omentum hydrogel for 3 days and stained with collagen (red), OCT4 (green) and KI67 (blue). Scale bar = 50 μm. I. SEM imaging of differentiated implant (day 30). Scale bar = 10 μm. J. Immunofluorescence of differentiated implant at day 30. Cells express motor neuron specific marker (HB9; blue), neuronal marker (TUJ1; green) and dendritic marker (MAP2; red). Scale bar = 50 μm. K. Immunofluorescence of differentiated implants at day 30. Cells express synaptic (SYP; blue), neuronal intermediate filament (NFM; green) and dendritic (MAP2; red) markers. Scale bar = 50 μm. L. Heatmap of RNA-seq expression Z-scores calculated at days 0, 20 and 30 of differentiation. [Figure 2-2] Same as above [Figure 2-3] Same as above [Figure 3-1]FIG. 3A-J are photographs and graphs showing ECM content and cellular function of implants. A. Heatmap of RNA-seq expression Z-scores calculated for 17 secreted ECM proteins at differentiation days 0, 20, and 30. B. Bar graph showing enriched function at differentiation day 30 (top 500 genes) associated with the 17 ECM genes in (A). C-E. Expression of ECM-associated proteins (NTN1 and SLIT1). TUJ1 is used to identify neurons. Scale bar = 50 μm. C. iPSC implant prior to differentiation. D. iPSC-derived spinal cord neurons differentiated on MATRIGEL™ for 30 days. E. iPSC-derived spinal cord implant at differentiation day 30. F. Rheological properties of acellular hydrogels and implants at differentiation days 0, 15, and 30. G. Neurite outgrowth from implants at day 30, cultured for 72 hours on MATRIGEL™ coated surfaces. Scale bar = 50 μm. H. Neurite branching networks formed among the three implants after 72 hours. Scale bar = 250 μm. I. Calcium response of the implants to depolarization with KCl. J. Calcium response of the implants to glutamate stimulation. [Figure 3-2] Same as above [Figure 3-3] Same as above [Diagram 3-4] Same as above [Figure 4-1]Figure 4, A-P, illustrates the acute SCI model. A. Schematic of the study. Mice were subjected to hemisection at T10, leaving the left hind limb paralyzed. Treatments were performed immediately after the induction of injury. Mice were allowed to survive for 3 months, during which time scar analysis, catwalk gait analysis and anterograde tracking were performed. After 2 weeks of tracking, mice were transcardially perfused and cords were extracted for analysis. B. Treatments performed: three control groups, termed "untreated" (animals treated with saline), "cells" (animals treated with iPSCs derived from SC neurons in suspension), "hydrogel" (animals treated with an acellular omentum-based hydrogel), and an experimental group, termed "implant" (animals treated with iPSCs derived from SC neuroimplants). C-G. Cell analysis of the lesion site 7 days after treatment. C. Engraftment of the implant 7 days after implantation. Before implantation, cells were labeled with cytopainter (red). D. Representative images of microglial cell (IBA1) expression in the "implant" group. E. Quantification of IBA1 density in the various groups. F. Representative images of astrocytes (GFAP) in the implant group. G. Quantification of GFAP expression in the various groups. H-K: Cell analysis of the lesion site after 12 weeks of treatment. H. Representative images of neural stem cells (NESTIN) and neurons (TUJ1) in the "implant" group. I. Quantification of TUJ1 density in the various groups. J. Quantification of NESTIN density in the various groups. K. Representative images of ECM molecules (NTN1 and SLIT1). L. Montage of anterograde tracing in the "implant" group. Yellow arrows indicate axons observed caudal to the lesion site. M. Quantification of axons at various distances from the lesion, labeled by anterograde tracing. N-O. Catwalk gait analysis parameters after 12 weeks of treatment. n. Degree of normal step sequence pattern (regularity index). o. Maximum pressure exerted by the left hind limb (Left hind limb maximum strength mean value). P. Weight of mice after 12 weeks of treatment. All scale bars = 100 μm. [Figure 4-2] Same as above [Figure 4-3] Same as above [Figure 4-4] Same as above [Figure 5-1]Figure 5, A-M, illustrates the chronic SCI model. A. Schematic of the study. Mice were subjected to hemisection at T10 while leaving the left hindlimb paralyzed. After 6 weeks, the lesion site was re-exposed, the scar was excised, and treatment was performed intracavitarily. Mice were allowed to survive for an additional 8 weeks after treatment, during which MRI and behavioral studies were performed. After 8 weeks of treatment, the cord was excised for histological analysis. B. Coronal T2W MRI of the spinal cord 5 weeks after the initial SCI (1 week before scar resection). The yellow arrow indicates the complete hemisection performed on the left side of the spinal cord. C-F. Diffusion tensor images 4 weeks after treatment. C. Glyph-based visualization of the diffusion tensor shown on the background of the axial diffusion tensor image. Blue indicates fibers in the rostral-caudal axis, red indicates left-right direction both medial and lateral, and green indicates anterior-posterior direction. D. Fiber tractography reconstructed in the axial plane. Red fibers are ipsilateral to the initial hemisection, green fibers are contralateral (intact). Left panels for each treatment are frontal views and right panels are lateral views. E. Percentage of fibers passing through the lesion, normalized to the healthy side. F. Fractional anisotropy (FA) measurements. G-J. Quantification of protein expression in the lesion site 8 weeks after treatment. G. GFAP expression density. H. IBA1 expression density. I. TUJ1 expression density. J. Number of GAP43 positive cells. K-M. Behavioral studies performed during the recovery phase (post-treatment). K. Regularity index of the catwalk step sequence. L. Maximum pressure applied to the left hind paw. M. Grid walking test: correct steps by the injured paw out of all attempted steps. *p<0.05 was detected only in the implant and untreated groups. **p<0.05 was detected between the implant and all control groups. [Figure 5-2] Same as above [Figure 5-3] Same as above [Figure 5-4] Same as above [Figure 6] Figure 6, A-B, are photographs showing nuclear staining of native omentum (left) and decellularized omentum (right) under the same exposure conditions. Scale bar = 200 μm. [Figure 7] FIG. 7 is a histogram of fiber diameters of omentum-based hydrogels. [Figure 8]Figure 8 shows a table of 17 ECM genes and their associated enriched functions. Purple indicates that the gene is associated with an enriched function. Bars indicate the number of ECM genes associated with a particular function. [Figure 9] FIG. 9 is a graph showing the complex viscosity at various frequencies of acellular hydrogels and implants at days 0, 20, and 30 of differentiation. [Figure 10] Figure 10 is a photograph showing cell engraftment of injected cells 7 days after treatment. Cells were labeled with cytopainter (red) prior to transplantation. Nuclei are shown in blue (Hoechst). Scale bar = 100 μm. [Figure 11] Figure 11 is a representative image of immunolabeling of the lesion site in the acute model, 7 days after treatment. Scale bar = 100 μm. The top panel is microglial cells (IBA1), the middle panel is astrocytes (GFAP), and the bottom panel is neuronal nuclei (NeuN shown in red) and astrocytes (GFAP shown in green). [Figure 12] Figure 12 is a graph showing the number of proliferative astrocytes. Double positive staining for Ki67 proliferative marker and GFAP (astrocytes). [Figure 13] Figure 13 is a photograph showing immunostaining of neural stem cells (NESTIN) and neurons (TUJ1) in the lesion site of acutely treated animals 12 weeks after implant treatment. Scale bar = 100 μm. [Figure 14] Figure 14: Photograph of anterograde tracing. Photomontage of anterograde tracing with TMRD. Yellow arrows indicate axons at or caudal to the lesion site. Scale bar = 50 μm. [Figure 15] Figure 15 is a glyph-based visualization of diffusion tensor images after scar resection and 1 week after treatment. Glyphs are displayed on the background of the axial diffusion tensor images. Blue indicates fibers in the rostral-caudal axis, red indicates left-right direction both medial and lateral, and green indicates anterior-posterior direction. [Figure 16]Figure 16: Fiber tractography 4 weeks after scar resection and treatment. Fiber tractography reconstructed in the axial plane. Red fibers are ipsilateral to the initial hemisection and green fibers are contralateral (intact). Fibers are shown en face with axial slices through the lesion epicenter, rostral (+0.6 mm above the lesion) and caudal (-0.6 mm below the lesion). [Figure 17] Figure 17 is a photograph showing the cellular content in the chronic phase after 8 weeks of treatment. Immunostaining of astrocytes (GFAP), microglial cells (IBA1), nerve growth associated protein (GAP43) and neurons (TUJ1) in the lesion area. Scale bar = 100 μm. [Figure 18] Figure 18A-B are graphs showing the results of behavioral studies 4 weeks after chronic scar resection and treatment: A. Grid walking testing sensorimotor activity in mice. B. Left hindlimb maximum strength correlated to pressure exerted by the mouse on the injured paw. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present invention, in some embodiments thereof, relates to spinal cord injury, and more particularly to compositions for treating chronic spinal cord injury.

[0036] Before describing at least one embodiment of the present invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by way of examples. The invention is capable of other embodiments and of being practiced or carried out in various ways.

[0037] Traumatic spinal cord injury (SCI) has an immediate and devastating impact on motor control and all aspects of a patient's health and quality of life. The initial trauma causes direct damage, often leading to cell death, disruption of the blood-spinal cord barrier, and degradation of the extracellular matrix (ECM). These processes initiate a secondary inflammatory injury cascade, causing progressive chronic tissue damage and resulting in the formation of a glial scar.

[0038] We have now found a method for the treatment of injured spinal cord (SC) in the chronic phase. Using an approach that recapitulates the embryonic development of SC, we fabricated ECM-based particles (also referred to herein as mini-implants) that provide the initial support and interactive material for the culture and proliferation of iPSCs. Throughout the in vitro culture stage, the cells of the particles and the ECM exhibited a synergistic effect that mimicked the spinal cord formation process in the embryo. The ECM-based particles supported the initiation of efficient cell differentiation in 3D by providing the cells with a suitable microenvironment. During cell differentiation, the cells continuously remodeled the fibers of the particles by secreting specific motor neuron ECM proteins, providing an inductive microenvironment for cell-cell and cell-substrate interactions. Overall, this dynamic microenvironment supplied distinct biochemical cues for different developmental stages and promoted the construction of functional SC implants.

[0039] We then investigated the potential of implants (i.e., multiple ECM-based particles) to treat injured SCs in the chronic phase. At this stage, the scar is fully developed and spontaneous behavioral recovery has reached a plateau. After resection of the scar tissue and insertion of an implant to fill the resection gap formed after scar removal, the anatomy and tissue morphology were assessed by MRI, showing strong anisotropy and a large number of intact nerve fibers within the SC (Figure 5B). Analysis of the cellular content at the lesion site showed reduced levels of inflammation and a larger number of neurons with increased expression of markers associated with axonal sprouting during development and regeneration (Figure 5E-J). These results translate into a significantly higher level of functional recovery as judged by sensorimotor functional analysis (Figure 5K-M).

[0040] Thus, according to a first aspect of the present invention there is provided a composition comprising a plurality of fibrous particles produced from decellularized omentum, the fibrous particles having a diameter of between 750 microns and 3 mm, the fibrous particles comprising a network of mature neurons.

[0041] The phrase "fibrous particles" refers to non-liquid particles made from decellularized omentum fibers that contain collagen and / or elastin fibers.

[0042] The fibrous particles described herein act as a scaffold for the creation of neural networks.

[0043] As used herein, the term "scaffold" refers to a three-dimensional structure comprising a biocompatible material that provides a suitable surface for cell attachment and growth. The scaffold can further provide mechanical stability and support.

[0044] The decellularized omentum fibers contained in the particles typically have a diameter of 50-200 nm, more typically 50-150 nm, and more typically 60-120 nm.

[0045] The particles of this aspect of the invention are typically circular, and more particularly substantially spherical, e.g., spherical, elliptical, semi-spherical, hemispherical, irregular spheres having flat or concave or convex portions, semi-elliptical, irregular elliptical having flat or concave or convex portions.

[0046] The particles typically have an average diameter of from 750 microns to 3 mm, more typically from 1 to 2 mm.

[0047] According to certain embodiments, the particles have an average diameter of greater than 500 microns.

[0048] As used herein, the term "particle size" refers to a particle size measured, for example, by a laser scattering particle size distribution measuring device.

[0049] As used herein, the phrase "decellularized omentum" refers to the extracellular matrix (ECM) that supports omentum tissue architecture and is therefore free of cellular components due to having undergone a decellularization process (i.e., removal of all cells from the tissue).

[0050] Decellularized omentum contains extracellular matrix (ECM) components.

[0051] As used herein, the phrase "extracellular matrix (ECM)" refers to the complex network of materials produced by the cells of a tissue and secreted into the surrounding extracellular space and / or medium, which typically work in conjunction with the cells of the tissue to give the tissue its mechanical and structural properties. In general, the ECM includes fibrous elements (particularly collagen, elastin, and / or reticulin), cell adhesion polypeptides (e.g., fibronectin, laminin, and / or cell adhesion glycoproteins), and space-filling molecules (usually glycosaminoglycans (GAGs), proteoglycans).

[0052] The omentum may be harvested from mammalian species such as humans, pigs, cows, goats, etc. After tissue harvest, the tissue can be placed in 0.9% saline for immediate processing or stored for later use, preferably at a temperature of about -20°C to about -80°C.

[0053] According to one embodiment of the present invention, the decellularization comprises: (a) exposing the omentum to a hypotonic solution; (b) dehydrating the omentum after step (a); and (c) after step (b), extracting fat from the dehydrated omentum using polar and non-polar extractants; (d) after step (c), rehydrating the dehydrated omentum; (e) removing the cells from the rehydrated omentum after step (d); This is carried out by.

[0054] A hypotonic solution is one in which the concentration of electrolytes is lower than the concentration inside the cell. In this situation, osmotic pressure attempts to equalize the electrolyte concentrations inside and outside the cell wall, causing water to move into the cell.

[0055] Preferably, the hypotonic buffer used by the method according to this aspect of the invention is a 10 mM Tris solution having a pH of about 8.0 and containing about 0.1% (w / v) EDTA (5 mM EDTA).

[0056] The hypotonic buffer may contain additional agents such as serine protease inhibitors (eg, phenylmethanesulfonyl fluoride or phenylmethylsulfonyl fluoride (PMSF)) and / or anionic detergents such as sodium dodecyl sulfate (SDS).

[0057] According to this aspect of the invention, the tissue is exposed to a hypotonic buffer for a period of time that results in a biological effect, namely cell swelling and bursting.

[0058] After hypotonic shock, the tissue may optionally be subjected to a freeze-thaw cycle.

[0059] The freeze-thaw process preferably involves freezing the tissue, e.g., at −10 to −80° C., typically at −80° C., for 2 to 24 hours, followed by thawing the tissue for about 2, 3 or 4 hours to room temperature or above (e.g., 37° C.) This process is carried out at least once, and preferably two or three times, in the presence of a hypotonic buffer.

[0060] Dehydration involves treating the omentum with one or more dehydration solvents, where such one or more treatments of the omentum are performed with the dehydration solvent and / or an aqueous solution of this solvent. The one or more treatments may be successive steps in a method performed with solutions having different ratios of dehydration solvent to water, such as gradually reducing the water in the solution with each successive treatment, and the final treatment may involve the use of a pure solvent, i.e., a solvent that is not an aqueous solution.

[0061] The dehydration solvent may be a low molecular weight organic solvent. In one embodiment, the dehydration solvent is one or more alcohols such as those selected from the group consisting of methanol, ethanol, isopropanol, propanol, and combinations thereof.

[0062] According to certain embodiments, the omentum is dehydrated by rinsing once in 70% ethanol (eg, for 10-60 minutes) and two or three times in 100% ethanol, each for 10-60 minutes.

[0063] After dehydration, fat may be extracted from the omentum using at least one polar and one non-polar solvent, which may be done in one or more extraction steps.

[0064] Examples of non-polar solvents are non-polar organic solvents such as hexane, xylene, benzene, toluene, ethyl acetate and combinations thereof. Polar solvents useful for the extraction solvent include acetone, dioxane, acetonitrile and combinations thereof. In one embodiment, the extraction solvent is selected from acetone, hexane, xylene and combinations thereof. Non-polar solvents include, for example, hexane, xylene and combinations thereof.

[0065] Fat extraction may be performed in a fat extraction step by contacting the dehydrated omentum with an extraction solvent for various periods of time.

[0066] Preferably, the polar lipids of the tissue are extracted by washing with a polar extractant (e.g., 100% acetone) for 10-60 minutes. This may be repeated several times (e.g., three times). The non-polar lipids may then be extracted by incubation for about 24 hours with a mixture of non-polar agent:polar agent (e.g., 60 / 40 (v / v) hexane:acetone solution (three changes) or 60 / 40 (v / v) hexane:isopropanol solution (three changes)).

[0067] After fat extraction, the defatted omentum is optionally rehydrated. The defatted omentum may be rehydrated by contacting the defatted omentum with a rehydration solvent, such as an alcohol or aqueous alcohol solution, for example, an alcohol solution containing about 60% to about 70% alcohol. Low molecular weight alcohols, such as methanol, ethanol, isopropanol, propanol, and combinations thereof, may be used.

[0068] The delipidated omentum is then decellularized. Any decellularization process known to those skilled in the art may be applied to decellularize the delipidated omentum.

[0069] Exemplary methods for decellularizing the omentum are described in U.S. Patent No. 20150202348 and WO 2014 / 037942, the contents of which are incorporated herein by reference.

[0070] In one embodiment, the delipidated omentum may be decellularized by solubilization of nuclear and cytoplasmic components. For example, the delipidated omentum may be immersed in a decellularization buffer, such as a buffer containing a non-ionic surfactant and a metal salt dissolved in acid, for a period of time, typically at least about 30 minutes. Non-ionic surfactants useful in the present invention include polysorbates such as TWEEN 80, ethoxylated alcohols such as TRITON® X-100, polyethanols such as HP 40 and IGEPAL CA-630, and combinations thereof. Metal salts that may be used include magnesium chloride, magnesium phosphate, magnesium acetate, and magnesium citrate, and combinations thereof, which are typically dissolved in Tris-HCl.

[0071] According to another embodiment, the delipidated omentum may be decellularized by enzymatic proteolytic digestion to digest the cellular components in the tissue while leaving the ECM components (e.g., collagen and elastin), thereby obtaining a matrix that exhibits the mechanical and structural properties of the ECM of the native tissue. It will be appreciated that measures must be taken to digest the cellular components of the tissue while leaving the ECM components. These measures are described further below and include, for example, adjusting the concentration of active ingredients (e.g., trypsin) in the digestion fluid and adjusting incubation times.

[0072] The proteolytic digestion according to this aspect of the invention can be carried out using various proteolytic enzymes.Non-limiting examples of suitable proteolytic enzymes include trypsin and pancreatin, available from various sources, such as Sigma (St. Louis, Missouri, USA).According to a preferred embodiment of this aspect of the invention, the proteolytic digestion is carried out using trypsin.

[0073] Digestion with trypsin is preferably performed at a trypsin concentration of 0.01-0.25% (w / v), more preferably 0.02-0.2% (w / v), more preferably 0.05-0.1 (w / v), and even more preferably about 0.05% (w / v). For example, in order to efficiently digest all cellular components of the tissue, a trypsin solution containing 0.05% (w / v) trypsin (Sigma), 0.02% (w / v) EDTA and antibiotics (penicillin / streptomycin, 1000 units / ml and 0.1 mg / mL, respectively), pH=7.2 may be used.

[0074] Preferably, the tissue pieces are gently agitated (eg, at about 150 rpm) while immersed in the digestive fluid to ensure complete penetration of all cells of the tissue with the digestive fluid.

[0075] It should be noted that the concentration of the digestion solution and the incubation time therein are dependent on the size of the tissue pieces utilized, and one skilled in the art can adjust the conditions depending on the desired size and type of tissue.

[0076] Preferably, the tissue pieces are digested for at least 1 hour, although digestion may be carried out for up to 24 hours.

[0077] After decellularization, the omentum may optionally be defatted again (eg, using a combination of polar and non-polar solvents).

[0078] The method according to this aspect of the invention optionally and preferably includes an additional step of obtaining nucleic acid-free tissue by removing nucleic acids (and residual nucleic acids) from the tissue. As used herein, the phrase "nucleic acid-free tissue" refers to tissue that is free of more than 99% of any nucleic acid or fragments thereof as measured using conventional methods (e.g., spectrophotometry, electrophoresis). Such a step utilizes a deoxyribonuclease solution (optionally also a ribonuclease solution). Suitable nucleases include deoxyribonucleases and / or ribonucleases (20 μg / ml solution in Hank's Balanced Salt Solution (HBSS), Sigma, Beit Haemek, Israel) or a combination of both (e.g., Benzonase). High concentrations of salts such as sodium chloride, from 0.5 M to 3 M, can also be used to remove nucleic acids.

[0079] Cellular components are then typically removed from the tissue. Removal of digested components from the tissue can be achieved using various washing solutions, such as detergent solutions (e.g., ionic and non-ionic detergents such as SDS, Triton X-100, Tween-20, Tween-80, etc.), available from, for example, Sigma (St. Louis, Missouri, USA) or Biolab (Atarot, Israel), Merck (Germany).

[0080] Preferably, the detergent solution used by the method according to this aspect of the invention comprises TRITON-X-100 (available from Merck & Co.) For efficient removal of all digested cellular components, TRITON-X-100 is provided in a concentration range of 0.05-2.5% (v / v), more preferably 0.05-2% (v / v), more preferably 0.1-2% (v / v), even more preferably 1% (v / v).

[0081] Optionally, the detergent solution also contains ammonium hydroxide, which, together with TRITON-X-100, aids in the disruption and lysis of cell nuclei, cytoskeletal proteins and membranes.

[0082] Preferably, ammonium hydroxide is provided at a concentration of 0.05-1.5% (v / v), more preferably at a concentration of 0.05-1% (v / v), even more preferably at a concentration of 0.1-1% (v / v) (e.g. 0.1%).

[0083] The concentrations of TRITON-X-100 and ammonium hydroxide in the detergent solution may vary depending on the type and size of tissue being processed, and one of skill in the art can adjust such concentrations depending on the tissue used.

[0084] Incubation of the tissue (or tissue fragment) with the detergent solution can last from a few minutes to a few hours or even days depending on the type and size of the tissue and the concentration of the detergent solution used, and such incubation time can be adjusted by a person skilled in the art. Preferably, incubation with the detergent solution is carried out for at least 1 hour. According to one embodiment, 1 to 4 cycles of incubation with the detergent solution are carried out until no bubbles are observed.

[0085] The detergent solution is preferably removed by washing the substrate several times (eg, at least three times) with water or saline until there is no evidence of detergent solution in the substrate.

[0086] Optionally, the decellularized ECM is then sterilized. Sterilization of the decellularized ECM may be performed using methods known in the art. In one embodiment, the decellularized omentum is contacted with the disinfectant solution for a time period effective to disinfect the decellularized omentum, for example, at least about 0.5 hours, typically about 1 hour to about 12 hours. The decellularized omentum may be fully immersed in the disinfectant solution. The disinfectant solution may include an alcohol or an alcohol-water solution, and may also include an acid. The disinfectant solution may include one or more of the following: ethanol, methanol, isopropanol, propanol, hydrogen peroxide, peracetic acid, and combinations thereof. In one embodiment, the disinfectant solution includes ethanol, for example, a 70% ethanol solution. Optionally, the decellularized omentum may be washed one or more times with ultrapure water.

[0087] After washing and optional sterilization, the decellularized tissue may be dehydrated, for example by freeze-drying.

[0088] Other methods contemplated by the inventors for decellularizing tissue include those described in U.S. Pat. Nos. 4,776,853, 4,801,299, and U.S. Patent Publication No. 20090163990, the contents of each of which are incorporated herein by reference in their entireties.

[0089] The decellularized omentum of this embodiment of the present invention typically contains less than 20% cells compared to the amount of cells in the omentum before decellularization, more preferably contains less than 15% cells compared to the amount of cells in the omentum before decellularization, more preferably contains less than 10% cells compared to the amount of cells in the omentum before decellularization, more preferably contains less than 5% cells compared to the amount of cells in the omentum before decellularization, and more preferably contains less than 2% cells compared to the amount of cells in the omentum before decellularization.

[0090] In one embodiment, the decellularized omentum is free of cellular components.

[0091] As used herein, the phrase "free of cellular components" refers to free of greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% (e.g., 100%) of the cellular components present in naturally occurring (e.g., native) omentum.

[0092] As used herein, the phrase "cellular components" refers to cell membrane components or intracellular components that constitute a cell. Examples of cellular components include cellular structures (e.g., organelles) or molecules contained therein. Such examples include, but are not limited to, cell nuclei, nucleic acids, residual nucleic acids (e.g., fragmented nucleic acid sequences), cell membranes and / or residual cell membranes (e.g., fragmented membranes) present within cells of a tissue. It will be understood that such decellularized matrices cannot induce an immune response when implanted into a subject, since all cellular components have been removed from the tissue.

[0093] The decellularized omentum of this aspect of the invention is essentially lipid-free, and the inventors have found that the degree of lipid extraction from the tissue correlates with the ability to induce cell attachment, maintain cell viability, and promote proper organization of cells into the tissue.

[0094] As used herein, the phrase "lipid-free" refers to a composition that contains less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of the lipids present in natural (e.g., native) omentum.

[0095] To generate particles made from the decellularized omentum, the decellularized omentum is solubilized.

[0096] Solubilization of decellularized ECM may be performed as described in Freytes et al., Biomaterials 29 (2008) 1630-1637 and US Patent Application No. 20120156250, the contents of which are incorporated herein by reference.

[0097] Typically, to effect solubilization of the decellularized omentum, the decellularized omentum is first dehydrated (eg, lyophilized).

[0098] The lyophilized decellularized omentum may be cut into small pieces (e.g., crushed) or ground into a powder and then subjected to a second proteolytic digestion. The digestion is carried out under conditions that allow the proteolytic enzymes to digest and solubilize the ECM. Thus, according to one embodiment, the digestion is carried out in the presence of an acid (e.g., hydrochloric acid) to a pH of about 1-4.

[0099] Proteolytic digestion according to this aspect of the invention can be carried out using a variety of proteolytic enzymes.Non-limiting examples of suitable proteolytic enzymes include trypsin, pepsin, collagenase and pancreatin, available from a variety of sources, such as Sigma (St. Louis, Missouri, USA), and combinations thereof.Matrix degrading enzymes, such as matrix metalloproteases, are also contemplated.

[0100] It should be noted that the concentration of the digestive fluid and the incubation time therein will depend on the type of tissue being processed and the size of the tissue pieces utilized, and one skilled in the art will be able to adjust the conditions depending on the desired size and type of tissue.

[0101] Preferably, the tissue pieces are incubated for at least about 20 hours, more preferably at least about 24 hours. Preferably, the digestive fluid is changed at least once so that the total incubation time in the digestive fluid is at least 40-48 hours.

[0102] After the decellularized omental ECM has been solubilized, the pH of the solution is raised (e.g., to about pH 7) to irreversibly inactivate the proteolytic enzymes. The decellularized and solubilized omentum may be stored at this stage at a low temperature below 20° C., e.g., 4° C., with the decellularized ECM remaining in solution.

[0103] The solubilized decellularized omentum can form a gel at temperatures greater than about 30°C, greater than about 31°C, greater than about 32°C, greater than about 33°C, greater than about 34°C, greater than about 35°C, greater than about 36°C, or greater than about 37°C.

[0104] To make the particles of this aspect of the invention, the solubilized decellularized omentum is typically mixed with stem cells that can differentiate into neurons to form neural networks.

[0105] Below is a description of various stem cells that can be used to generate particles according to this aspect of the invention.

[0106] The stem cells may or may not be genetically modified. For example, the cells may be genetically modified to express an exogenous polypeptide or polynucleotide (e.g., an RNA silencing agent such as an siRNA).

[0107] As used herein, the term "stem cell" refers to a cell (e.g., totipotent, pluripotent or multipotent stem cell) that can remain in an undifferentiated state in culture for an extended period of time until induced to differentiate into other cell types (e.g., fully differentiated cells) with specific specialized functions. Totipotent cells, such as embryonic cells within the first few cell divisions after fertilization, can differentiate into embryonic and extraembryonic cells and are the only cells that can differentiate into viable human beings. Preferably, the term "pluripotent stem cell" refers to a cell that can differentiate into all three germ layers, i.e., ectoderm, endoderm and mesoderm, or remain in an undifferentiated state. Pluripotent stem cells include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Multipotent stem cells include adult stem cells and hematopoietic stem cells.

[0108] The phrase "embryonic stem cells" refers to embryonic cells that can differentiate into cells of all three germ layers (i.e., endoderm, ectoderm and mesoderm) or can remain undifferentiated. The phrase "embryonic stem cells" may include cells obtained from pre-implantation embryonic tissue (e.g., blastocysts) formed after conception (i.e., pre-implantation blastocysts), expanded blastocyst cells (EBCs) obtained from blastocysts at the post-implantation / pre-gastrulation stage (see WO 2006 / 040763), embryonic germ (EG) cells obtained from fetal reproductive tissue at any time during pregnancy, preferably before the 10th week of pregnancy, and cells derived from unfertilized eggs derived by parthenogenesis (parthenogenetic eggs).

[0109] Induced pluripotent stem cells (iPSCs, embryonic-like stem cells) are cells obtained by dedifferentiation of adult somatic cells endowed with pluripotency (i.e. the ability to differentiate into the three embryonic germ cell layers, i.e. endoderm, ectoderm and mesoderm). According to some embodiments of the invention, such cells are obtained from differentiated tissues (e.g. somatic tissues such as the omentum) and dedifferentiated by genetic manipulations that reprogram the cells to acquire embryonic stem cell characteristics. According to some embodiments of the invention, induced pluripotent stem cells are generated by inducing the expression of Oct-4, Sox2, Kfl4 and c-Myc in omental cells.

[0110] The phrase "adult stem cells" (also called "tissue stem cells" or somatic tissue-derived stem cells) refers to any stem cell derived from somatic tissue [of postnatal or prenatal animals, particularly humans]. Adult stem cells are generally considered to be multipotent stem cells that can differentiate into multiple cell types. Adult stem cells can be obtained from any adult, neonatal or fetal tissue, such as adipose tissue, skin, kidney, liver, prostate, pancreas, intestine, bone marrow and placenta.

[0111] Hematopoietic stem cells, also referred to as adult tissue stem cells, include stem cells obtained from blood or bone marrow tissue of individuals of any age, or from umbilical cord blood of newborn individuals. According to this aspect of some embodiments of the invention, the preferred stem cells are embryonic stem cells, preferably embryonic stem cells of human or primate (e.g. monkey) origin.

[0112] Placental stem cells and umbilical cord blood stem cells may be referred to as "juvenile stem cells."

[0113] Embryonic stem cells of some embodiments of the present invention can be obtained using well-known cell culture methods. For example, human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo preimplantation embryos or in vitro fertilized (IVF) embryos. Alternatively, single-cell human embryos can be allowed to progress to the blastocyst stage. For isolation of human ES cells, the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by immunosurgery, which lyses and removes trophectoderm cells from the intact ICM by gentle pipetting. The ICM is then plated in tissue culture flasks containing an appropriate medium that allows proliferation. After 9-15 days, the ICM-derived outgrowths are dissociated into clumps by mechanical dissociation or enzymatic digestion, and the cells are then replated in fresh tissue culture medium. Colonies exhibiting undifferentiated morphology are individually selected with a micropipette, mechanically dissociated into clumps, and replated. The resulting ES cells are routinely dissociated every 4-7 days. For further details on methods for preparing human ES cells, see Thomson et al. [U.S. Pat. No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92: 7844, 1995], Bongso et al. [Hum Reprod 4: 706, 1989], and Gardner et al. [Fertil. Steril. 69: 84, 1998].

[0114] It will be appreciated that commercially available stem cells may also be used according to some embodiments of the present invention. Human ES cells can be purchased from the NIH Human Embryonic Stem Cell Registry [http: / / grants.nih.gov / stem_cells / registry / current.htm]. Non-limiting examples of commercially available embryonic stem cell lines include BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE32, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, HUES1, HUES2, HUES3, HUES4, HUES5, HUES6, HUES7, HUES8, HUES9, HUES10, HUES11, HUES12, HUES13, HUES14, HUES15, HUES16, HUES17, HUES18, HUES19, HUES20, HUES21, HUES22, HUES23, HUES24, HUES25, HUES26, HUES27, HUES28, HUES29, HUES30, HUES31, HUES32, HUES33, HUES34, HUES35, HUES36, HUES37, HUES38, HUES39, HUES40, HUES41, HUES42, HUES43, HUES44, HUES45, HUES46, HUES47, HUES48, HUES49, HUES50, HUES51, HUES52, HUES53, HUES54, HUES55, HUES56, HUES57, HUES58, HUES59, HUES60, HUES61, HUES62, HUES63, HUES64, HUES65, HUES66, HUES67, HUES68, HUES69 S26, HUES27, HUES28, CyT49, RUES3, WA01, UCSF4, NYUES1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, UCLA1, UCLA2, UCLA3, WA077(H7), WA09(H9), WA13(H13), WA14(H14), HUES62, HUES63, HUES64, CT1, CT2, CT3, CT4, MA135, Eneavour-2, WIBR1, WIBR2, WIBR3, WIBR4, WIBR5, WIBR6, HUES45, Shef3, Shef6, BJNhem19, BJNhem20, SA001, and SA001.

[0115] Furthermore, ES cells have been successfully used in mouse (Mills and Bradley, 2001), golden hamster (Doetschman et al., 1988, Dev Biol. 127: 224-7), rat (Iannaccone et al., 1994, Dev Biol. 163: 288-92), rabbit (Giles et al. 1993, Mol Reprod Dev. 36: 130-8; Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424-33), and several livestock species (Notarianni et al., 1991, J Reprod Fertil Suppl. 43: 255-60; Wheeler 1994, Reprod Fertil Dev. 6: 563-8; Mitalipova et al., 2001, Cloning. 3: 59-67] and other species, including non-human primate species (rhesus monkeys and marmosets) [Thomson et al., 1995, Proc Natl Acad Sci USA. 92: 7844-8; Thomson et al., 1996, Biol Reprod. 55: 254-9].

[0116] Expanded blastocyst cells (EBCs) can be obtained from blastocysts at a pre-gastrulation stage, at least 9 days post-fertilization. Prior to culturing the blastocysts, the zona pellucida is digested (e.g., by Tyrode's acid solution (Sigma-Aldrich, St. Louis, MO, USA)) to expose the inner cell mass. Blastocysts are then cultured in vitro as whole embryos at least 9 days post-fertilization but no later than 14 days (i.e., prior to the gastrulation event) using standard embryonic stem cell culture methods.

[0117] Another method for preparing ES cells is described in Chung et al., Cell Stem Cell, Volume 2, Issue 2, 113-117, 7 February 2008. This method involves the removal of a single cell from an embryo during an in vitro fertilization process. The embryo is not destroyed during this process.

[0118] EG cells are prepared from primordial germ cells obtained from fetuses of about 8-11 weeks gestation (for human fetuses) using laboratory techniques known to those skilled in the art. The genital ridge is dissociated, cut into small pieces, and then separated into cells by mechanical dissociation. EG cells are then grown in tissue culture flasks using appropriate medium. The cells are cultured with daily changes of medium until cell morphology consistent with EG cells is observed, typically after 7-30 days or until passages 1-4. For further details of methods for the preparation of human EG cells, see Shamblott et al. [Proc. Natl. Acad. Sci. USA 95: 13726, 1998] and U.S. Patent No. 6,090,622.

[0119] Non-fertilized eggs (parthenogenetic eggs) derived from embryonic stem cells (e.g., human ESCs) by parthenogenesis are known in the art (e.g., Zhenyu Lu et al., 2010. J. Assist Reprod. Genet. 27:285-291; "Derivation and long-term culture of human parthenogenetic embryonic stem cells using human foreskin feeders", which is incorporated herein by reference in its entirety). Parthenogenesis refers to the initiation of cell division by activation of an egg in the absence of sperm cells, e.g., using electrical or chemical stimulation. The activated egg (parthenogenetic egg) can develop into a primitive embryonic structure (called a blastocyst), but the cell cannot develop to the extent that it is referred to as pluripotent, i.e., it cannot develop the extraembryonic tissues (such as amniotic fluid) required for a viable human fetus.

[0120] According to certain embodiments, iPSC cells are reprogrammed (ie, de-differentiated) from omental stromal cells.

[0121] Adult tissue stem cells are described by Alison, MR [J Pathol. 2003 200(5): 547-50], Cai, J. et al. [Blood Cells Mol Dis. 2003 31(1): 18-27], Collins, AT et al. [J Cell Sci. 2001; 114(Pt 21): 3865-72], Potten, CS and Morris, RJ [Epithelial stem cells in vivo. 1988. J. Cell Sci. Suppl. 10, 45-62], Dominici, M et al. [J. Biol. Regul. Homeost. Agents. 2001, 15: 28-37], Caplan and Haynesworth [US Pat. No. 5,486,359], Jones EA et al. [Arthritis Rheum. 2002, 46(12): 3349-60]. Embryonic stem cells can be isolated using a variety of methods known in the art, such as those disclosed in Eventov-Friedman S, et al., PLoS Med. 2006, 3: e215; Eventov-Friedman S, et al., Proc Natl Acad Sci USA. 2005, 102: 2928-33; Dekel B, et al., 2003, Nat Med. 9: 53-60; and Dekel B, et al., 2002, J. Am. Soc. Nephrol. 13: 977-90. Hematopoietic stem cells can be isolated using a variety of methods known in the art, such as those disclosed in "Handbook of Stem Cells", edited by Robert Lanze, Elsevier Academic Press, 2004, Chapter 54, pp 609-614, isolation and characterization of hematopoietic stem cells, by Gerald J Spangrude and William B Stayton.

[0122] In general, the isolation of adult tissue stem cells is based on the discrete sites (or niches) of each cell type contained in the adult tissue stem cells, i.e., stem cells, transiently proliferating cells, and terminally differentiated cells [Potten, CS and Morris, RJ (1988). Epithelial stem cells in vivo. J. Cell Sci. Suppl. 10, 45-62]. Thus, adult tissue stem cells, for example prostate tissue, are digested with collagenase and subjected to repeated unit gravity centrifugation to separate the prostate epithelial structures (e.g., organoids, acini, and ducts) from the stromal cells. The organoids are then disaggregated into a single cell suspension by incubation with Trypsin / EDTA (Life Technologies, Paisley, UK) and CD44-positive basal stem cells are isolated from the terminally differentiated CD57-positive luminal secretory cells by labeling with anti-human CD44 antibody (clone G44-26) (Pharmingen, Becton Dickinson, Oxford, UK) and incubation with Goat anti Mouse IgG microbeads from MACS (Miltenyi Biotec Ltd, Surrey, UK). The cell suspension is then applied to a MACS column, and the basal cells are eluted and resuspended in WAJC404 complete medium [Robinson, EJ et al. (1998). Basal cells are progenitors of luminal cells in primary cultures of differentiating human prostatic epithelium Prostate 37, 149-160].

[0123] Basal stem cells can attach to basement membrane proteins more rapidly than other basal cells [Jones, PH et al. (1995). Stem cell patterning and fate in human epidermis. Cell 60, 83-93; Shinohara, T., et al. (1999). β1- and α6-integrin are surface markers on mouse spermatogonial stem cells. Proc. Natl. Acad. Sci. USA 96, 5504-5509], CD44-positive basal cells were cultured in Dulbecco's phosphate-buffered saline (PBS) (Oxoid Ltd, Basingstoke, UK) containing 0.3% bovine serum albumin (fraction V, Sigma-Aldrich, Poole, UK), collagen type I (52 μg / ml), collagen type IV (88 μg / ml), or laminin 1 (100 μg / ml) (Becton The cells are plated onto tissue culture dishes coated with Dickinson's Biocoat®. After 5 minutes, the tissue culture dishes are washed with PBS and the adherent cells, including prostate tissue basal stem cells, are harvested with trypsin-EDTA.

[0124] BM-derived stem cells, mesenchymal stem cells In one embodiment, the stem cells utilized by some embodiments of the present invention are BM-derived stem cells, including hematopoietic stem cells, stromal stem cells, or mesenchymal stem cells (Dominici, M et al., 2001. Bone marrow mesenchymal cells: biological properties and clinical applications. J. Biol. Regul. Homeost. Agents. 15: 28-37). BM-derived stem cells may be obtained from the iliac crest, femur, tibia, spine, rib, or other medullary cavities.

[0125] Among the above BM-derived stem cells, mesenchymal stem cells are formative pluripotent blast cells. Mesenchymal stem cells can give rise to one or more mesenchymal tissues (e.g., adipose tissue, bone tissue, cartilage tissue, elastic connective tissue, fibrous connective tissue, myoblasts) as well as tissues other than those derived from embryonic mesoderm (e.g., nervous system cells) depending on various influences from bioactive factors such as cytokines. Although such cells can be isolated from embryonic yolk sac, placenta, umbilical cord, fetal skin, adolescent skin, blood and other tissues, their abundance in BM far exceeds their abundance in other tissues, and therefore isolation from BM is currently preferred.

[0126] Methods for isolating, purifying and expanding mesenchymal stem cells (MSCs) are known in the art and include, for example, those disclosed by Caplan and Haynesworth in U.S. Pat. No. 5,486,359 and by Jones EA et al., 2002, Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells, Arthritis Rheum. 46(12): 3349-60.

[0127] Preferably, mesenchymal stem cell cultures are generated by diluting BM aspirate (usually 20 ml) with an equal volume of Hank's Balanced Salt Solution (HBSS) (GIBCO Laboratories, Grand Island, NY, USA) and layering over approximately 10 ml of a Ficoll column (Ficoll-Paque) (Pharmacia, Piscataway, NJ, USA). After centrifugation at 2,500×g for 30 min, the mononuclear cell layer is removed from the interface and suspended in HBSS. The cells are then centrifuged at 1,500×g for 15 min and resuspended in complete medium (MEM, alpha medium without deoxyribonucleotides or ribonucleotides, GIBCO), 20% fetal calf serum (FCS) from a lot selected for rapid proliferation of MSCs (Atlanta Biologicals, Norcross, GA), 100 units / ml penicillin (GIBCO), 100 μg / ml streptomycin (GIBCO), and 2 mM L-glutamine (GIBCO). The resuspended cells are plated in approximately 25 ml of medium in a 10 cm culture dish (Corning Glass Works, Corning, NY) and incubated at 37° C. under 5% humidified CO2. After 24 hours of culture, non-adherent cells are discarded and adherent cells are washed extensively twice with phosphate-buffered saline (PBS). The medium is replaced with fresh complete medium every 3 or 4 days for approximately 14 days. The adherent cells are then harvested using 0.25% trypsin and 1 mM EDTA (Trypsin / EDTA, GIBCO) for 5 min at 37°C, replated onto 6 cm plates, and cultured for an additional 14 days. The cells are then trypsinized and counted using a cell counting device, for example a hemocytometer (Hausser Scientific, Horsham, PA). Cultured cells are harvested by centrifugation and titrated to 1-2 x 10 cells per ml using 5% DMSO and 30% FCS. 6 Resuspend the cells to a concentration of 1 ml. Slowly freeze aliquots of approximately 1 ml each and store in liquid nitrogen.

[0128] To expand the mesenchymal stem cell fraction, frozen cells are thawed at 37°C, diluted in complete medium, and harvested by centrifugation to remove DMSO. Cells are resuspended in complete medium and grown at approximately 5,000 cells / cm. 2 After 24 hours of culture, non-adherent cells are removed and adherent cells are harvested using Trypsin / EDTA and dissociated by passage through a fine Pasteur pipette, preferably at a concentration of about 1.5 to about 3.0 cells / cm. 2 Under these conditions, MSC cultures can be expanded for approximately 50 population doublings and expanded up to approximately 2000-fold [Colter DC., et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA. 97: 3213-3218, 2000].

[0129] The MSC cultures utilized by some embodiments of the present invention preferably contain three cell populations defined by their morphological characteristics: small agranular cells (hereinafter referred to as RS-1), small granular cells (hereinafter referred to as RS-2) and large intermediate granular cells (hereinafter referred to as mature MSCs). The presence and concentration of such cells in culture can be measured, for example, by identifying the presence or absence of various cell surface markers using immunofluorescence, in situ hybridization and activity assays.

[0130] When MSCs are cultured under the culture conditions of some embodiments of the present invention, they show negative staining for hematopoietic stem cell markers CD34, CD11B, CD43 and CD45. A small portion of the cells (less than 10%) are weakly positive for CD31 and / or CD38 markers. In addition, mature MSCs are weakly positive for hematopoietic stem cell marker CD117 (c-Kit), moderately positive for osteogenic MSC marker Stro-1 [Simmons, PJ & Torok-Storb, B. (1991). Blood 78, 5562], and positive for thymocyte and peripheral T lymphocyte marker CD90 (Thy-1). Meanwhile, RS-1 cells are negative for CD117 and Stro1 markers and weakly positive for CD90 marker, and RS-2 cells are negative for all these markers.

[0131] As mentioned above, to make the particles of the invention, the solubilized decellularized omentum is mixed with stem cells (e.g., dissociated colonies of iPSC cells). Droplets of 1-10 μL, e.g., 2-5 μL of solution may be created using a droplet forming device (e.g., a pipette) on a solid surface such as silicon glass or plastic. Other surface types are contemplated, including oil-based and aqueous surfaces. The droplets are formed at a temperature that maintains the decellularized omentum as a liquid. Once the droplets are formed, they are then exposed to a temperature above 30° C. (e.g., 37° C.) for at least half an hour to ensure that the droplets solidify and form solid, gel-like particles. After gelation, the particles are then cultured in a medium, and the cells seeded therein continue to survive.

[0132] In one embodiment, the droplets (or particles) are not exposed to a chemical crosslinker.

[0133] In another embodiment, the droplets (or particles) are exposed to an additional cross-linking agent.

[0134] Exemplary chemical crosslinkers such as carbodiimides (EDC and DCC), N-hydroxysuccinimide esters (NHS esters), imidoesters, maleimides, haloacetyls, pyridyl disulfides, hydrazides, alkoxyamines, aryl azides, diazirines, Staudinger reagent pairs, and the like are contemplated.

[0135] Alternatively or additionally, enzymes such as transglutaminase, sortase, laccase / peroxidase, lysyl oxidase / amine oxidase may be used for cross-linking. Other enzymes are disclosed in Heck et al., Appl Microbiol Biotechnol. 2013 Jan; 97(2): 461-475, the contents of which are incorporated by reference.

[0136] Additional chemical crosslinkers that may be used in the present invention include carbodiimides (EDC and DCC), N-hydroxysuccinimide esters (NHS esters), imidoesters, maleimides, haloacetyls, pyridyl disulfides, hydrazides, alkoxyamines, aryl azides, diazirines, and Staudinger's reagents.

[0137] Other methods of forming decellularized omentum particles are known in the art, including those disclosed in U.S. Patent Application No. 20180361023, the contents of which are incorporated herein by reference.

[0138] It will be appreciated that the cells are not seeded onto preformed particles, but rather are mixed into the decellularized omentum while it is in liquid form (i.e., prior to particle formation) and therefore are typically uniformly distributed throughout the particles.

[0139] Prior to the differentiation step, the stem cells contained in the particles may be allowed to grow to fill the volume of the particles, for example for at least 1 day, 3 days, 7 days or more. The particles are cultured in a medium that prevents differentiation of the cells (i.e. helps maintain the pluripotency of the cells). Typically, each particle contains about 15,000-150,000 stem cells at the start of the differentiation step. In one embodiment, the differentiation step is initiated when the cells reach about 90% confluence.

[0140] In one embodiment, the particles are cultured in a medium containing a neural cell differentiation agent under conditions that promote diffusion of the neural cell differentiation agent into the particles.

[0141] Methods for differentiating pluripotent stem cells into neural cells are known in the art, and are described in Edri et al., Advanced materials 31, 1803895 (2019); Shimojo, et al. Mol Brain 8, 79 (2015); Yi et al., Stem Cells International, 2018, Article ID 3628578; Faravelli et al. Stem Cell Research & Therapy, 2014, 5:87; Wada et al. PLoS One, 2009, Volum 4, Issue 8, e6722; Qu et al., Nature Communications,5:3449 | DOI: 10.1038 / ncomms4449; Karumayaram et al., Stem Cells. 2009 April; 27(4): 806-811. doi:10.1002 / stem.31, the contents of each of which are incorporated herein by reference.

[0142] Methods for differentiating mesenchymal stem cells into cells of the neural lineage are provided, for example, in WO 2006 / 134602, WO 2009 / 144718, WO 2007 / 066338 and WO 2004 / 046348, the teachings of which are incorporated herein by reference.

[0143] Exemplary neural cell differentiation agents that can be used in the differentiation process include, but are not limited to, retinoic acid, valproic acid and derivatives thereof (e.g., esters, salts, retinoids, retinates, valproates, etc.), thyroid hormone or other agonists of thyroid hormone receptors, noggin, BDNF, NT4 / 5 or other agonists of NTRK2 receptors, agents that increase expression of transcription factors ASCL1, OLIG1, d113 agonists, Notch1, 2, 3 or 4 antagonists, nicastrin, gamma secretase inhibitors including small molecule inhibitors of Aph1A, Aph1B, Psen1, Psen2 and PSENEN, delta-like ligand (D11)-1 antagonists, delta-like ligand (D11)-4, jagged1 antagonists, jagged2 antagonists, numb agonists or numb-like agonists.

[0144] According to one embodiment, the culturing is performed under conditions that promote differentiation of the cells within the particle into mature neurons (eg, mature motor neurons) that form neural networks within the particle.

[0145] The neurons may be excitatory or inhibitory neurons.

[0146] In one embodiment, the neuron comprises a motor neuron.

[0147] Neurons according to this aspect of the invention express markers indicative of mature neurons (e.g., express a dendritic marker such as MAP2, a synaptic marker (SYP), and a neuronal intermediate filament marker (NFM).

[0148] In another embodiment, the neurons express markers of mature motor neurons, including but not limited to, choline acetyltransferase (ChAT), HB9 (also known as MNX1), and ISL-1.

[0149] Preferably, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the cells of the neural network in the particle express markers of mature motor neurons as measured by immunofluorescence or flow cytometry analysis.

[0150] For example, in one embodiment, greater than 50%, 60%, 70%, 80%, 90% of the motor neurons in the particle express neuron-specific class III β-tubulin (TUJ1) as measured by flow cytometry.

[0151] According to another embodiment, greater than 50%, 60%, 70%, 80%, 90% of the motor neurons in the particles express motor neuron and pancreatic homeobox 1 (MNX1) as measured by flow cytometry.

[0152] In yet another embodiment, neurons in a network can be induced to fire synchronously in vitro.

[0153] The term "neural network" refers to a set of interconnected neurons that contain dendrites and have synapses between them. Neurons of a neural network in a single particle can interact (under appropriate conditions) with neurons of a neural network of another particle. In one embodiment, the neural network also includes neurofilaments.

[0154] Neurons of a network within a particular particle can connect with neurons of a network of another particle under appropriate conditions. In one embodiment, the connection between neurons of two particles is made after implantation at the site of injury. In another embodiment, the connection between neurons of two particles can be made ex vivo (see, e.g., FIG. 3, G and H).

[0155] In one embodiment, the differentiation process comprises: (a) Culturing iPSCs in the presence of an ALK5 inhibitor, an ALK2 / ALK3 inhibitor and a GSK3 inhibitor; (b) subsequent incubation in the presence of retinoic acid and a Hedgehog pathway agonist (purmorphamine); (c) Subsequent incubation in the presence of sonic hedgehog and retinoic acid; (d) subsequent incubation in the presence of neurotrophic factors (e.g., BDNF), ascorbic acid, a Hedgehog pathway agonist (purmorphamine), and retinoic acid; (e) Subsequent incubation in the presence of a γ-secretase inhibitor (e.g., DAPT) and Includes.

[0156] The particles may also include additional cells, such as astrocytes.

[0157] The particles can also be incorporated (e.g., attached, coated, embedded, impregnated) with therapeutic compounds or agents that modulate cell activity. Campbell et al. (US Patent Application No. 20030125410), which is incorporated by reference as if fully set forth herein, disclose a method for fabricating a 3D scaffold for stem cell proliferation, the scaffold having a preformed gradient of therapeutic compounds. According to Campbell et al., the scaffold material falls into the category of "bio-ink". Such "bio-ink" is suitable for use in the compositions and methods of the present invention.

[0158] Exemplary agents that may be incorporated into the particles of the invention include, but are not limited to, agents that promote cell adhesion (e.g., fibronectin, integrins), cell colony formation, cell proliferation, cell differentiation, anti-inflammation, cell extravasation and / or cell migration. Thus, for example, the agent may be an amino acid, a small molecule chemical, a peptide, a polypeptide, a protein, DNA, RNA, a lipid and / or a proteoglycan.

[0159] Proteins that may be incorporated into the particles of the present invention include, but are not limited to, extracellular matrix proteins, cell adhesion proteins, growth factors, cytokines, hormones, proteases and protease substrates. Thus, exemplary proteins include vascular endothelial-derived growth factor (VEGF), activin-A, retinoic acid, epidermal growth factor, bone morphogenetic proteins, TGFβ, hepatocyte growth factor, platelet-derived growth factor, TGFα, IGF-I and II, hematopoietic growth factors, heparin-binding growth factors, peptide growth factors, erythropoietin, interleukins, tumor necrosis factors, interferons, colony-stimulating factors, basic and acidic fibroblast growth factors, nerve growth factors (NGF) or muscle morphogenetic factors. The particular growth factor used must be appropriate for the desired cellular activity. The regulatory effects of the large family of growth factors are well known to those skilled in the art.

[0160] In one embodiment, only neural cells are contained in the particle.

[0161] In yet another embodiment, the particles are essentially free of pluripotent stem cells.

[0162] Preferably, at least 50%, 60%, 70%, 80%, 90% of the pluripotent stem cells are differentiated into a neural cell lineage, more preferably into a mature neural cell type.

[0163] Thus, for example, no more than 1%, 3%, 5%, 10%, 15% or 20% of cells express pluripotency markers (eg, TRA-1-60, SSEA4, OCT4) as measured by flow cytometry or immunohistochemistry.

[0164] The particles of the present invention may be used by themselves in the treatment of spinal cord injury or may be mixed with suitable carriers or excipients and used as part of a pharmaceutical composition.

[0165] As used herein, a "pharmaceutical composition" refers to a formulation of one or more particles described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to a living organism.

[0166] Hereinafter, the terms "physiologically acceptable carrier" and "pharmaceutical acceptable carrier", which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to the living body and does not impair the biological activity and properties of the administered compound. Adjuvants are included in these terms.

[0167] Examples of carriers include, but are not limited to, propylene glycol, saline, emulsions, buffers, culture media such as DMEM or RPMI, cryopreservation media containing components that scavenge free radicals and provide pH buffering, oncotic / osmolarity maintenance, energy substrates, and ionic concentrations that balance intracellular conditions at low temperatures, as well as mixtures of organic solvents and water. Typically, pharmaceutical carriers maintain particle numbers (e.g., do not reduce by more than 90%) and maintain viability of cells within the particles in the composition for at least 24 hours, at least 48 hours, or even at least 96 hours.

[0168] According to certain embodiments, the physiologically acceptable carrier is saline.

[0169] As used herein, the term "excipient" refers to an inactive substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0170] Techniques for drug formulation and administration are described in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA (latest edition), which is incorporated herein by reference.

[0171] Pharmaceutical compositions suitable for use in the context of some embodiments of the present invention include compositions in which the active ingredient is contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount refers to an amount of the active ingredient (particles as described herein) effective to prevent, reduce or ameliorate symptoms of disease or injury (e.g., spinal cord injury) or prolong the survival of the subject being treated.

[0172] Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0173] For any preparation used in the method of the present invention, the therapeutically effective amount or dose can be estimated first from in vitro and cell culture assays.For example, a dose can be formulated in an animal model to achieve a desired concentration or titer.Such information can be used to more accurately determine a useful dose in humans.

[0174] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical methods in vitro in cell cultures or experimental animals. The data obtained from these in vitro assays, cell culture assays, and animal studies can be used in formulating a range of dosages for use in humans. Dosages may vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition (see, for example, Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.1).

[0175] Dosage and interval may be adjusted individually to provide a level of active ingredient sufficient to induce or inhibit a biological effect (minimal effective concentration (MEC)). The MEC varies from formulation to formulation but can be estimated from in vitro data. The dose required to achieve the MEC depends on individual characteristics and route of administration. Detection assays can be used to measure plasma concentrations.

[0176] Depending on the severity and responsiveness of the condition to be treated, administration may be single or multiple and the duration of treatment may range from several days to several weeks, or until a cure is effected or a diminution of the pathology is achieved.

[0177] The amount of composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician.

[0178] The particles of the present invention, at least in some embodiments, may be prepackaged in a unit dosage form in a ready-to-use syringe. The syringe may be labeled with the name of the particle and its source. The labeling may also include information related to the function of the particle. The syringe may be packaged in a package that is also labeled with information about the particle.

[0179] The particles described herein are useful for treating spinal cord injuries.

[0180] Thus, according to another aspect of the present invention, there is provided a method of treating chronic spinal cord injury in a subject, comprising implanting a composition described herein at the site of injury in the subject at least three months after spinal cord injury, thereby treating the spinal cord injury.

[0181] As used herein, the phrase "spinal cord injury" refers to damage to the spinal cord caused by trauma, not disease. Symptoms can vary widely, for example from pain to paralysis to incontinence, depending on where the spinal cord and nerve roots are damaged. Spinal cord injuries are described in various levels of "incompleteness," which can vary from not affecting the patient at all to "complete" damage, i.e., total loss of function. Spinal cord injuries have many causes, but are typically associated with major trauma from car accidents, falls, sports injuries, and violence. The abbreviation "SCI" ​​stands for spinal cord injury.

[0182] Spinal cord injury can be affected by secondary tissue damage, including but not limited to glial scarring, myelin inhibition, demyelination, cell death, lack of neurotrophic support, ischemia, free radical formation and excitotoxicity. This secondary tissue damage typically occurs at least 3, 4, 5, 6 months or more after the initial injury. This stage is also called chronic spinal cord injury.

[0183] According to certain embodiments, the particles are implanted at the injury site after removal of scar tissue. The particles are typically implanted using a device suitable for administration of the particles. In one embodiment, the device is a syringe (e.g., a syringe with an inner diameter of 1-5 mm). After injection of the particles at the injury site, the carrier is typically sucked back into the syringe.

[0184] As used herein, the term "about" refers to ±10%.

[0185] The terms "comprises," "comprising," "includes," "including," "having" and their cognates mean "including but not limited to."

[0186] The term "consisting of" means "including and limited to."

[0187] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0188] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0189] Throughout this application, various embodiments of the present invention may be described in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as indefinitely limiting the scope of the present invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as the individual numbers within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed the individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6, as well as the subranges 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc. This applies regardless of the breadth of the range.

[0190] Whenever a numerical range is given herein, it is meant to include any cited numbers (decimals or integers) within the stated range. The phrases "range / between" a first and second numerical value and "to" and "range / from" a first numerical value to a second numerical value are used interchangeably herein and are meant to include the first and second numerical values ​​and all decimals and integers therebetween.

[0191] As used herein, the term "method" refers to methods, means, techniques and procedures for accomplishing a given task, including but not limited to those methods, means, techniques and procedures known to practitioners in the chemical, pharmacological, biological, biochemical and medical fields, or readily developed from methods, means, techniques and procedures known to practitioners in the chemical, pharmacological, biological, biochemical and medical fields.

[0192] It will be understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or in any other described embodiment of the invention, as appropriate. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0193] Various embodiments and aspects of the present invention as described in detail herein and claimed below are provided with experimental support in the following examples. EXAMPLES

[0194] Reference will now be made to the following examples which, together with the above descriptions, illustrate some embodiments of the invention in a non-limiting manner.

[0195] General references are provided throughout this document. The procedures in the references are believed to be well known in the art and are provided for the convenience of the reader. All information contained in the references is incorporated herein by reference.

[0196] Example 1 material and method Formation of omentum hydrogel: Decellularization of omentum (M. Shevach et al., Biomedical materials 10, 034106 (2015)): Porcine omentum (Kibbutz Rahab, Israel) was washed with phosphate-buffered saline (PBS) to remove major blood vessels. The samples were then transferred to a hypotonic buffer (10 mM Tris, 5 mM ethylenediaminetetraacetic acid (EDTA) and 1 μM phenylmethanesulfonyl fluoride (PMSF), pH 8.0) for 1 h. The tissue was then frozen and thawed three times using the same buffer. The tissue was washed progressively with 70% ethanol and 100% ethanol for 30 min each. Lipids were extracted by washing three times for 30 min with 100% acetone, followed by incubation (three changes) in a 60 / 40 (v / v) hexane:acetone solution for 24 h. The delipidated tissue was washed with 100% ethanol for 30 minutes and incubated in 70% ethanol overnight at 4°C. The tissue was then washed four times with PBS (pH 7.4) and incubated overnight in 0.25% trypsin-EDTA (Biological Industries). The tissue was washed extensively with PBS and incubated for 24 hours (three changes) in 1.5 M NaCl, followed by 1 hour in 50 mM Tris (pH 8.0), 1% Triton-X100 (Sigma) solution. The decellularized tissue was washed with PBS, followed by two washes with distilled water, then frozen (-20°C) and lyophilized.

[0197] Solubilization of omentum hydrogel: After lyophilization, the decellularized omentum was ground into a powder (Wiley Mini-Mill) (Thomas Scientific, Swedesboro, NJ). The dried ground omentum was solubilized with pepsin (4000 U mg -1 (Sigma) at 1 mg ml -1 The enzymatic digestion was carried out in the solution at room temperature for 96 hours. The pH was then adjusted to 7.4 using DMEM / F12×10 or PBS×10 (Biological Industries). The final concentration of decellularized omentum in the titration solution was 1.5% (w / v). At least 10 porcine omentum were used.

[0198] Culture of undifferentiated iPSCs: iPSCs were generated from omental stromal cells. Undifferentiated cells were cultured on culture plates precoated with MATRIGEL™ (BD, NJ) diluted to 250 μg / mL in DMEM / F12 (Biological Industries, Beit Haemek, Israel) or cultured within omental hydrogels. All cells were cultured at 37°C and 5% CO2. Undifferentiated iPSCs were maintained in NutriStem® (Biological Industries) medium containing 0.1% penicillin / streptomycin (Biological Industries). Medium was changed daily and cells were passaged weekly using 1 U / mL dispase (Stemcell Technologies, Vancouver, Canada) followed by mechanical disruption. iPSCs were seeded into small colonies in the presence of Y-27632 (10 μM) (Tocris, UK).

[0199] Creation and differentiation of spinal motor neuron implants: Dissociated iPSC colonies were mixed in equal volumes with 1.5% mesh-based hydrogel. A pipette was used to create 3 μL droplets. The implants were crosslinked for 30 min at 37 °C on a damp towel, after which medium was added. Undifferentiated cells were cultured in Nutristem, which was changed daily, until they reached 90% confluence. Cells were differentiated as previously described (R. Edri et al., Advanced materials 31, 1803895 (2019)). Briefly, after reaching approximately 90% confluence, the medium was changed to Knockout / DMEM supplemented with 15% Knockout Serum, 0.1% penicillin / streptomycin, 0.5% l-glutamine, 1% non-essential amino acids (Invitrogen), 10 μM β-mercaptoethanol, 10 mM SB-431542 (Tocris), 1 μM LDN-193189 (Tocris), and 3 μM CHIR-99021, and gradually changed to DMEM / F12 supplemented with N2 every 3 days (on the 3rd day, it was 3 / 4 Knockout / DMED and 1 / 4 DMEM / F12 supplemented with N2 only in the F12 part, and on the 6th day, it was changed to 1 / 2 and 1 / 2). On days 4 and 6, 1 μM retinoic acid and 1 μM purmorphamine (Tocris) were added to the motor neuron medium. On day 8, DMEM F / 12 supplemented with N2, 30 ng / mL sonic hedgehog (R&D) and 1 μM retinoic acid was added to the cells (1 / 3 of the final volume, without changing the medium). From day 10 onwards, the medium was changed to DMEM / F12 supplemented with N2, 0.1% P / S, 5 μg / mL BDNF (R&D), 200 μM ascorbic acid (Sigma), 1 μM purmorphamine (Tocris) and 1 μM retinoic acid. From day 15 onwards, 5 μM DAPT (Tocris) was also added and the concentration of purmorphamine was reduced to 500 nM. The medium was changed every 3 days until the 30th day.

[0200] Immunostaining and confocal imaging: Cell implants were fixed with 4% formaldehyde, permeabilized with 0.05% (v / v) Triton X-100, blocked with PBS, 1% bovine serum albumin (BSA), 10% fetal bovine serum (FBS), and stained with the indicated primary antibodies followed by secondary antibodies (listed in the antibody list (Supporting Information)). Cells and implants were imaged using an upright confocal microscope (Nikon, ECLIPSE NI-E) and an inverted fluorescent microscope (Nikon, ECLIPSE TI-E). Images were processed and analyzed using NIS elements software (Nikon Instruments).

[0201] Neurite outgrowth assay: For the neurite outgrowth assay, implants at day 30 of differentiation were placed on plates coated with 250 μg / mL MATRIGEL™. The constructs were cultured for 3 days, then fixed with 4% formaldehyde and imaged using an inverted fluorescent microscope (Nikon, ECLIPSE TI-E).

[0202] RNA-seq and bioinformatics analysis: RNA samples from implants at days 0, 20 and 30 were extracted using the miRNeasy kit (Qiagen, Hilden, Germany) and treated with DNase (Qiagen). Pooled samples were quantified in at least two different experimental replicates. Library quality control was performed using FASTQC (version 0.11.5), followed by quality and adapter trimming using Cutadapt, version 1.1 (M. Martin, EMBnet. journal 17, 10-12 (2011)). All reads were aligned to the Homo sapiens reference genome using the TopHat aligner (Trapnell) with a maximum mismatch parameter of 3 and minimum and maximum intron sizes of 70 and 500000, respectively. Raw expression levels were calculated using HTseq-count, version 0.6.1 (S. Anders, PT Pyl, W. Huber, bioinformatics 31, 166-169 (2015)). Reads per kilobase million (RPKM) values ​​were then calculated based on the raw expression levels using annotations from the Ensembel gene reference file (version GRCh38.87).

[0203] Based on the fold change of RPKM in engineered spinal cord tissues (normalized to undifferentiated iPSCs at day 30 of differentiation), the 500 protein-coding genes with the highest increased expression were analyzed for functional enrichment using Ingenuity® Pathway Analysis (IPA®, Qiagen Bioinformatics, Redwood City, CA) software. For ECM analysis, 17 ECM-related genes were selected from the above 500 and functional enrichment was performed. The functions selected for ECM analysis were associated with at least 2 of the 17 ECM genes. The Z-score of each sample was calculated separately by RPKM value. The complete RNA data is available under the accession number GSE97341.

[0204] Calcium imaging: For calcium imaging, implants were incubated with 10 μM fluo-4AM (Invitrogen) and 0.1% Pluronic F-127 (Sigma-Aldrich) for 45 min at 37°C. Implants were then washed with Hank's buffered salt solution (HBSS) and imaged using an inverted fluorescence microscope (Nikon, Eclipse TI). Movies were acquired at 2 frames / sec using an ORCA-Flash 4.0 digital complementary metal-oxide semiconductor (CMOS) camera (Hamamatsu). HBSS was used as the external solution. A baseline was recorded for 30 s, after which depolarization was induced with KCl (50 mM, final concentration 25 mM) or glutamate (200 μM, final concentration 100 μM) solutions. Injections were performed in situ to measure cellular Ca 2+ Responses were recorded without interruption. Data were analyzed using ImageJ (NIH) and normalized by dividing each data set by the initial value (F / F0).

[0205] Spinal cord hemisection: Hemisection was performed as previously described (Y. Goldshmit et al., J Neurosci 24, 10064-10073 (2004)). Briefly, mice (20–30 g) were anesthetized by intraperitoneal injection of ketamine (100 mg / kg) and xylazine (16 mg / kg) in PBS. The spinal cord was exposed from low thoracic to high lumbar. After supination, a complete left hemisection was performed at T10, and the overlying muscle and skin were sutured. For acute injury, mice were treated immediately. Control groups were an untreated group treated with 10 μL saline, a cell group treated with dissociated iPSC-derived spinal cord neurons (differentiation day 30) suspended in 10 μL saline, and a hydrogel group treated with 0.75% crosslinked premembrane-based hydrogel. The implant group treated with differentiated iPSC-derived spinal cord neuron implants (day 30 of differentiation) was subjected to the test treatments.

[0206] In the chronic injury model, SCI was induced in the same way as in the acute injury model. Six weeks after the initial SCI, the animals were re-anesthetized and the lesion site was re-incised. The scar was carefully excised, and treatments were administered into the cavity formed by the excised scar. Mice were randomly assigned to four groups and allowed to survive from 1 week to 3 months after injury.

[0207] Tissue preparation and immunofluorescence labeling. After 1, 8, and 12 weeks of treatment, animals were anesthetized and perfused transcardially with 20 mL of PBS (pH 7.4) followed by 20 mL of 4% paraformaldehyde. Spinal cord tissue was dissected and post-fixed in 4% paraformaldehyde for 24 h at 4°C, and dehydrated with 20% v / v sucrose overnight at 4°C. Dissected tissue was embedded in OCT and cut longitudinally into 60 μm-thick frozen sections using a freezing microtome (Leica CM1950) (Germany).

[0208] For immunostaining, sections were permeabilized with 0.3% Triton X-100 in PBS solution, blocked with 10% FBS and 1% BSA in PBS for 1 h, and then incubated overnight at 4 °C with primary antibodies (antibody list in Supporting Information). After rinsing three times with PBS, sections were incubated with Alexa Fluor 488 / 594 / 647-labeled secondary antibodies for 1 h. Sections were then washed three times with PBS, counterstained with Hoechst 33528 (5 μg / mL) for 10 min at room temperature, then washed with PBS, left to dry, placed in antifade fluorescent mounting media and covered with glass slips. Sections were stored at 4 °C until analyzed using a Nikon confocal microscope. The integrated density of each of the injury epicenters was calculated automatically by ImageJ. For each animal, at least three sections near the injury epicenter (from dorsal to ventral) of the spinal cord were examined.

[0209] Anterograde axonal tracing. Axonal regeneration was examined using anterograde tracing (N=7 untreated, N=7 cells, N=10 hydrogels, and N=10 implants). Three months after SCI, the spinal cord was injected with tetramethylrhodamine dextran (TMRD, "Fluoro-Ruby", MW 10,000kD, Molecular Probes) at the level of the cervical enlargement ipsilateral to the lesion (Y. Goldshmit et al., J Neurosci 24, 10064-10073 (2004)). After 14 days, mice were perfused with PBS followed by 4% paraformaldehyde (PFA). Spinal cords were removed and postfixed in 4% cold PFA for 1 h followed by 20% sucrose in PBS overnight at 4°C. Longitudinal (horizontal) serial frozen sections were cut (60 μm) and slides were imaged using a fluorescence microscope (Nikon, ECLIPSE NI-E). Labeled axons within the white matter were quantified at 1000 μm, 500 μm, and 200 μm rostral to the lesion site and 100 μm caudal to the lesion site at 400x magnification. Photomontages of regenerating axons were made using a fluorescence microscope.

[0210] Catwalk gait analysis: Gait measurements were performed using a Catwalk XT system (Noldus Information Technology, The Netherlands). Data were transmitted to a computer and analyzed with CatWalk XT® software (version 10.6, Noldus). Each mouse was required to position itself on one side of the walkway and complete three compliant runs (variability <60%; time <5 s) to the other side. Coordination (regularity index) and the ability of the mouse to apply pressure to the injured paw (mean left hindlimb maximum strength) were tested. Parameters were calculated for each run and results were averaged for each animal at each time point.

[0211] Grid walking: One week before treatment and 1, 2, 4, 6, and 8 days after treatment, mice were tested by walking on a horizontal grid (grid spacing 1.2 × 1.2 cm, total area 35 × 45 cm). Each mouse was allowed to walk freely on the grid for 3 min. A misstep was counted when the left hind paw completely stepped off the grid with all toes and heel. The number of missteps and the total number of steps taken with the left hind paw were counted. Results were expressed as the percentage of correct steps with the left hind paw (Y. Goldshmit, Journal of Neurosurgery: Spine SPI 33, 692-704 (2020)).

[0212] Magnetic resonance imaging (MRI): MRI was performed 1 and 4 weeks after chronic scar resection using a Biospec 7T / 30 Scanner (Bruker) equipped with a 660mT / m gradient unit in a cross-coil configuration with an 86mm transmission volume coil and a 10mm loop coil as receiver. The untreated and cell groups had N=4, while the hydrogel and implant groups had N=5. Animals were anesthetized with 1-2% isoflurane in O2 on a heating pad, respiration was monitored, and body temperature was maintained at 37°C.

[0213] The MRI protocol included T2-weighted (T2w) images acquired with a Rapid Acquisition with Relaxation Enhancement (RARE) sequence and diffusion tensor images (DTI) acquired with a Diffusion-Weighted Spin-Echo Echo-Planar-Imaging pulse sequence (DW-SE-EPI). T2w acquisition was performed with the following parameters: TR = 8000 ms, effective TE: 30 ms, RARE factor 12 (repetition 3). 32 axial slices, 0.45 mm thickness (no gap), in-plane resolution 0.15 mm. 2 The whole brain was covered for 4 min with a 1000 s / mm2 DTI. DTI was performed for 5.5 min with the following parameters: TR / TE = 2500 / 19.2 ms, Δ / δ = 10 / 2.5 ms, EPI segments 2, gradient direction 30, b value 1000 s / mm2 2 , 3 B0 images, 30 axial slices, thickness 0.45 mm (no gap), in-plane resolution 0.30 mm 2 Total MRI protocol acquisition time was approximately 20 min. ExploreDTI software was used for DTI calculations and fiber tracking. Intrinsic components decomposed from the tensors were used to calculate fractional anisotropy maps. Regions of interest in the spinal cord were manually segmented in each slice. Fiber tracking was performed in the direction of the tracts with angles <30°, FA <0.15, and a resolution of 2 × 2 × 2.

[0214] Statistical analysis: All statistical analyses were performed using GraphPad Prism 8.00 (GraphPad Software, Inc.). Data are presented as mean ± SEM (standard error of the mean). Data were analyzed using Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. Values ​​were considered significantly different at p < 0.05.

[0215] Rheological properties: Rheological measurements (n=3; individual samples) were performed using a Discovery HR-3 hybrid Rheometer (TA Instruments, Delaware) with a parallel plate geometry of 8 mm diameter and equipped with a Peltier plate to maintain sample temperature. Samples were loaded at a temperature of 4°C and then increased to 37°C to induce gelation. During this time, samples were monitored at a fixed frequency of 1 Hz and strain of 1%. After gelation, the same samples were monitored using a frequency sweep (0.1-10 Hz) at strain of 1%. Cellular constructs were crosslinked prior to rheology and tested at different frequencies as previously described.

[0216] Scanning electron microscopy: To prepare samples (cellular or acellular), constructs were fixed in 2.5% glutaraldehyde (16–20 h at 4°C) followed by dehydration in a graded series of ethanol-water solutions (25–100%). All samples were critical point dried, mounted on aluminum stubs coated with conductive paint, and sputter coated with gold using a Polaron E 5100 coating apparatus (Quorum technologies, Lewes, UK). Samples were viewed on a JCM-6000PLUS NeoScope Benchtop (JEOL USA Inc., Peabody, MA).

[0217] Flow cytometry: For flow cytometry analysis, cells were isolated from implants using up to six cycles (30 min each) of enzymatic digestion with collagenase type II (95 U / mL; Worthington, Lakewood, NJ) and pancreatin (0.6 mg / mL; Sigma-Aldriin) in Dulbecco's modified Eagle's medium (DMEM, CaCl2·2H2O (1.8 mM), KCl (5.36 mM), MgSO4·7H2O (0.81 mM), NaCl (0.1 M), NaHCO3 (0.44 mM), NaH2PO4 (0.9 mM)). After each round of digestion, cells were centrifuged (120 g, 5 min), resuspended in DMEM / D12, and stored on ice. Cells were washed with PBS and then treated with Accutase (Stemcell Technologies, Vancouver, Canada) for 5 min at 37° C., followed by mechanical trituration to ensure dissociation into single cells.

[0218] For membrane proteins, cells were stained with labeled antibodies or isotype controls for 30 min at RT.

[0219] For intracellular proteins, cells were fixed with 4% formaldehyde, washed with PBS, permeabilized with 0.1% Triton, and incubated with primary and then secondary antibodies for 30 min each on ice. Cells were analyzed and data analysis was performed using a CytoFlex 4 flow cytometer (Beckman Coulter, USA). Positive populations were gated by unstained cells and appropriate isotype controls. At least three biological replicates were analyzed.

[0220] Antibody List Primary antibodies: OCT4 (ab27985, 1:100), Ki67 (ab16667, 1:250), TUJ1 (ab7751 / ab18207, 1:500), MAP2 (ab5392, 1:1000), NFM (ab24574, 1:1000), SYP (ab32127, 1:500), Iba1 (ab178846, 1:400), Nestin (ab134017; 1:2000), netrin1 (ab37390; 1:100), slit1 (ab115892; 1:100) and Cytopainter red (ab138893) were obtained from Abcam (Cambridge, MA). GFAP (Dako Z0334, 1:1000), NeuN (MAB377; 1:200; Millipore), Collagen I (MA1-26771, 1:2000), and TMRD (dextran, tetramethylrhodamine, D1817, 10000 MW) were obtained from Invitrogen. HB9 (81.5C10, 1:100) was purchased from DSHB.

[0221] Secondary antibodies: Alexa Fluor 488 (1:250; 111-545-003, Jackson), Alexa Fluor 555 (1:500; ab150118, Abcam), Alexa Fluor 647 (1:500; ab150135 / ab150175, Abcam). Nuclei were visualized with Hoechst 33258 (5 μg / mL).

[0222] Labeled antibodies: TRA-1-60-PE (1:100; 130-122-921, Miltenyi, Germany), SSEA-4 (1:100; 130-122-918, Miltenyi).

[0223] result Porcine omental tissue was decellularized while preserving the ECM (Figure 2A and B and Figure 6A-B). The omentum is an adipose tissue rich in blood vessels and sulfated glycosaminoglycans, and its ECM serves as a reservoir for stem cells in the body. The decellularized tissue was further processed into a thermoresponsive hydrogel (Figure 2C). This hydrogel exhibits weak mechanical properties at room temperature and is physically crosslinked under physiological conditions (Figure 2C and D).

[0224] During natural embryonic development, ECM fibers form a niche at the blastocyst stage to support stem cell renewal, differentiation, and morphogenesis. Scanning electron microscopy (SEM) images of the hydrogel revealed a fibrous structure with an average fiber diameter of 91.7 ± 33 nm (Figure 2E and Figure 7). During embryonic development, pluripotent stem cells proliferate within a confined microenvironment prior to differentiation. To mimic this physiological process, colonies of human iPSCs were mixed at low concentration within the meshwork hydrogel (Figure 2F). The particles were fabricated as described in the Materials and Methods section. The cells expressed high pluripotency markers (TRA-1-60, SSEA4, OCT4) and proliferation markers (KI67) within the hydrogel (Figure 2G and H), allowing them to proliferate and fill its volume. The iPSC implants (i.e., particles) were then subjected to a 30-day differentiation protocol within this 3D microenvironment to mimic the physiological process of neurogenesis. At day 30, the cells formed dense 3D networks throughout the particles (Figure 2I) and expressed general early and late neuronal markers such as TUJ1 and MAP2, as well as the specific motor neuron marker HB9 (Figure 2J). The formation of synapses and dendrites within the implants, as well as the formation of neurofilaments, indicated maturation of neural tissue (Figure 2K). Flow cytometry analysis showed that more than 85% of the cells expressed the neuronal marker TUJ1, and more than 60% were also positive for HB9. Furthermore, RNA sequencing at three different time points along the differentiation process (day 0, day 20, and day 30) revealed a downregulation of pluripotency-related genes and an upregulation of neuronal genes, particularly spinal motor neuron genes (Figure 2L). Multiple synergistic genes related to the functions of neuronal activity and maturation were significantly enriched.

[0225] During embryonic development, the dynamic ECM plays a crucial role in maintaining tissue-specific functions. Throughout the differentiation and maturation of the spinal cord, in addition to the initial matrix proteins provided by the fabricated hydrogel, the cells secreted additional specific ECM components and soluble factors that interact with the existing matrix. This remodeling of the ECM composition during development and differentiation provides a new microenvironment essential to support cell migration and promote axonal growth / guidance and synapse formation. To investigate this new extracellular microenvironment created during the differentiation process, neuronal ECM-related genes were studied. Among the 500 most elevated genes, the cells expressed 17 genes related to the production of essential ECM proteins that are not expressed in undifferentiated cells (Figure 3A). Further analysis revealed the involvement of those genes in enhanced functions related to neural tissue formation and function, including neurogenesis, axonal guidance, neurogenesis, neuronal branching, neuronal migration and neurotransmission (Figure 3B and Figure 8). These functions are essential for the proper formation of the spinal cord during embryonic development. 23The successful mimicking of these functions suggests that the supplied microenvironment and cells interact to form the proper 3D neural network. On 2D surfaces, the majority of soluble proteins are secreted into the medium, whereas in hydrogels they are trapped within the 3D microenvironment and can interact with the initial ECM to affect the encapsulated cells. To evaluate protein secretion and accumulation within the 3D microenvironment, cells differentiated on MATRIGEL™, undifferentiated implants, and 30-day implants were stained for SLIT1 and NTN1, representative ECM proteins that play a key role in axon guidance and are crucial for spinal cord positioning. SLIT1 protein prevents motor neuron migration towards the ventral floor plate, thereby allowing motor neurons to remain in the proper column. On the other hand, netrin is part of the larger laminin gene family and plays a key role in guiding axons to the midline. As shown, the protein was not detected in the day 0 implants nor on the 2D MATRIGEL™ surface, but was highly expressed in cells within the hydrogel (Figure 3C-E). Synthesis and presentation of ECM proteins influence the function of developing tissues. However, accumulation of ECM proteins within 3D hydrogels also altered the mechanical properties of the microenvironment. Although the complex viscosity of the acellular implant did not change over time, changes in the type and amount of ECM proteins significantly altered its biochemical content and increased the complex viscosity (Figure 3F and Figure 9).

[0226] To evaluate the ability of the implant to interact with the surrounding environment, isolated implants were seeded on a thin layer of MATRIGEL™, which demonstrated neurite outgrowth (Figure 3G). Furthermore, when multiple implants were placed at a distance of about 1 mm from each other, a branching network was formed between them within 3 days (Figure 3H). Such interactions between implants or between implants and healthy parts of tissue are crucial for efficient engraftment and for the initiation of regeneration processes in any neural tissue (Kawabata et al stem cell rep 2016). After confirming the formation of 3D networks, dendrites and synapses, calcium imaging was used to monitor the neuroelectrical activity of the implants. KCl is known to reliably depolarize neurons, allowing calcium ions to flow into the cells. As shown, KCl stimulation revealed a significant increase in fluorescence, indicating a chemical triggering effect (Figure 3I). Furthermore, a significant increase in calcium release could be induced by glutamate, an excitatory neurotransmitter (Figure 3J).

[0227] After engineering functional tissue implants that efficiently mimicked embryonic spinal cord development, their therapeutic potential was evaluated. First, as a proof of concept, we selected an acute injury model in mice. Here, a complete left hemisection was performed at T10 while the right side of the spinal cord was left intact (Figure 4A). Then, saline (untreated), a cell suspension in saline (cells), a cell-free hydrogel, or a complete implant (Figure 4B) were immediately inserted into the injury site and their ability to reduce inflammation and glial scar formation, promote neuroprotection and axonal regeneration, and improve the locomotion of mice was evaluated. To compare the engraftment of dissociated cells with that of a complete implant, we first pre-stained the cells with a fluorescent dye. One week after treatment, cells in the implant group were clearly detected at the lesion site, whereas cells applied in suspension were barely observed, highlighting the importance of a supportive microenvironment (Figure 4C and Figure 10). Following the breakdown of the blood-spinal cord barrier and hemorrhage, secondary injury occurs and immune cells from the periphery, or microglia from within the tissue, migrate towards the injury site. This inflammatory environment leads to further neuronal death and a massive accumulation of reactive astrocytes that form a glial scar. This process prevents further damage from spreading, but natural regeneration is inhibited. To evaluate the effect of the implants on inflammatory cell populations within the injury site, spinal cords were removed on day 7, sectioned, and stained for microglia (Iba1) and astrocyte (GFAP) markers. As shown, both the hydrogel and the implant significantly reduced the accumulation of both cell types (Figure 4C-G and Figure 11). Furthermore, the astrocytes detected within these groups were less reactive, as they also expressed Ki67 (Figure 12).

[0228] The reduced inflammation levels observed at day 7 led to a more permissive environment, resulting in significantly higher numbers of neurons (TUJ1) and neural stem cells (NESTIN) detected at week 12 in animals treated with the implant (Figure 4H-J and Figure 13). The latter are progenitor cells that have the capacity to differentiate into neurons or glial cells. Overall, cells at the lesion site were organized in the spinal tract direction and survived the injury (Figure 4H). This could be attributed to the high levels of guidance molecules observed within the surrounding microenvironment (Figure 14). Overall, such an organization of cells and essential ECM proteins could promote rewiring and regeneration throughout the injured spinal cord.

[0229] To assess the ability of the implant to transfer signals through the lesion site, mice were injected with an anterograde tracer molecule (TMRD) at the cervical level ipsilateral to the injury. Animals were maintained for an additional 2 weeks to allow the tracer to travel downstream through active neuronal axons. As shown, implant-treated mice had a significantly higher number of axons that reached and passed through the lesion site, allowing regrowth past the scar, whereas hydrogel-treated mice had a lower number of detected axons and none were observed with the other treatments (Figures 4K and L and Figure 15).

[0230] The presence of functional axons along the spinal tract is essential for proper motor function. Therefore, we next sought to evaluate the ability of the implant to improve the gait of treated mice by catwalk gait analysis. As shown, all animals partially recovered motor function, which is probably due to the ability of the animals to use the central pattern generator. However, recovery was significantly improved in animals treated with the implant (Fig. 4M and N). Compared to the untreated group, only animals treated with the implant showed significantly better coordinated movements, as judged by a higher regularity index (Fig. 4M). Similarly, the left hindlimb maximum strength, which indicates the ability of the mouse to apply pressure to the injured paw, was significantly higher only in animals treated with the implant (Fig. 4N). The improvement of these behavioral parameters may be due to the synergistic effect of reduced inflammation and the presence of neurons at the lesion site, which are essential for regeneration. This recovery led to a significantly higher increase in body weight (Fig. 4O), indicating another aspect of the improved overall condition.

[0231] Having demonstrated the ability of the implant to repair the injured spinal cord in the acute phase, we next evaluated its ability to regenerate tissue in a more clinically relevant model. A cascade of secondary events, including hemorrhage and edema, occurs immediately after the initial trauma. Therefore, we sought to evaluate the ability of the implant to treat the injured spinal cord once the injury has reached the chronic phase, when scarring is complete and spontaneous behavioral recovery has reached a plateau.

[0232] For this, a complete hemisection was performed as described for the acute phase. Six weeks after the initial SCI, the spinal cord was excised from the scar and the same procedure was performed within this cavity (Figure 4B). Structural, biochemical, cellular and behavioral parameters were then evaluated (Figure 5A).

[0233] As shown, complete hemisection was easily detectable by T2-weighted MRI before scar resection (Figure 5B). To visualize the repair process of the spinal tract, MRI with diffusion tensor imaging (DTI) was performed 1 week and 4 weeks after treatment. Diffusion anisotropy, which provides valuable information about the white matter of the spinal cord, is determined by both the structural orientation of the axons and the state of myelin. The diffusion tensor of each voxel of the MRI image is represented by the principal eigenvector that indicates the main direction of the fibers in that voxel. As shown, 1 week after scar resection, the main diffusion direction was random, not aligned along the spinal cord axis (Figure 16), which may indicate severe damage. However, at this stage of damage, some of the detected damage may have been caused by edema after scar resection, which may be fully or partially reversible. Analysis after 4 weeks of treatment showed a significant improvement in animals treated with the implant, as judged by the main diffusion direction (shown in blue) (Figure 5C). This improvement could be attributed to the incorporation of the implant and its ability to connect healthy axons above and below the lesion site. Streamline tractography was then used to visualize white matter fibers, their structural integrity, and damage to their fiber bundles. Reconstructed neuronal tracts showed pathological changes in the spinal cord at the lesion site in all animals (Figure 5D). However, tractography of untreated animals, or animals treated with cells in suspension, revealed higher displacement, deformation, and destruction at the lesion. In these animals, only a small amount of tissue or axons crossed the lesion site, and there was also gradual Wallerian degeneration above the scar resection level. In contrast, animals treated with the implant showed better preservation or recovery of damaged tracts, as judged by the number of tracts crossing the lesion site (Figure 5D and Figure 17). The amount of ipsilateral nerve fibers crossing the lesion site was quantified and compared to fibers at the same level in the healthy side. As shown, implant-treated mice had significantly higher axonal survival and / or regrowth through the lesion compared to untreated and cell-treated animals (Figure 5E). We next analyzed the fractional anisotropy, which depends on the water diffusivity in the extracellular space along the axon.Compared to all other treatments, animals treated with the implant demonstrated significantly higher values ​​at the lesion site (Figure 5F), indicating greater anisotropy and a greater number of intact nerve fibers.

[0234] The cellular content of the injury site was analyzed after a longer recovery period. The extensive injury caused by the removal of scar tissue did not further insert the support material, leaving a substantial cavity in some of the untreated animals. These spinal cords could not be excised and processed in one piece, which did not allow quantification of the cellular content at the injury site of these animals and reliable analysis of this group. Comparison of cell, hydrogel and implant treatments revealed that chronic inflammation was significantly reduced in animals treated with implants, as evidenced by a lower density of reactive astrocytes (Figure 5G and Figure 18A-B). Microglia were also significantly reduced in both hydrogel and implant treatments compared to the cell only group (Figure 5H and Figure 18A-B). As shown, a significantly higher number of neurons was observed in both hydrogel and implant treated mice (Figure 5I and Figure 18A-B). However, higher expression of GAP43, a marker associated with axonal sprouting in developing and regenerating spinal cord, was detected only in implant-treated animals (Fig. 5J and Fig. 18A-B), which may suggest active axonal sprouting at the lesion site that may result in gaps in healthy axons on either side of the lesion.

[0235] The ability of the implant to promote functional recovery was verified by sensory-motor function. During the experiment, the mice were subjected to behavioral studies including the Catwalk gate analysis and the grid walking test. The coordinated movement of the mice, represented by the step sequence regularity index, improved over time and reached its maximum capacity 6 weeks after treatment (Fig. 5K). Furthermore, in the implant-treated animals, higher pressure (on the injured paw) was already detected 1 week after implantation and was maintained throughout the experiment (Fig. 5L). Finally, motor and sensory recovery was observed in these animals, as shown by fewer missed steps on the injured paw in the grid walking analysis (Fig. 5M). The significant recovery detected in the sensory-motor of the implant-treated animals is in line with the DTI results that revealed intact fibers passing through the injury site (Fig. 5D).

[0236] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0237] It is the intention of the applicant that all publications, patents, and patent applications referenced herein are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference when referenced. Furthermore, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting. Additionally, the priority documents of this application are incorporated herein by reference in their entirety.

Claims

1. A composition comprising a plurality of fibrous particles made from decellularized omentum, said fibrous particles having a diameter of 750 microns to 3 mm, said fibrous particles comprising a network of mature neurons.

2. The composition of claim 1 , wherein the fibrous particles are essentially spherical.

3. The composition of claim 1 , wherein the mature neurons comprise motor neurons.

4. 4. The composition of claim 3, wherein greater than 50% of the motor neurons express neuron-specific class III β-tubulin (TUJ1) as measured by flow cytometry.

5. 4. The composition of claim 3, wherein greater than 50% of the cells express motor neuron and pancreatic homeobox 1 (MNX1) as measured by flow cytometry.

6. 2. The composition of claim 1, wherein the average diameter of the fibers of the fibrous particles is 50 to 200 nm.

7. The composition of claim 1 , wherein the omentum comprises human omentum.

8. The composition of claim 1 for treating a chronic spinal cord injury in a subject.

9. (i) the composition of claim 1; and (ii) a device for delivering the composition to the spinal cord of a subject; and Including, goods.

10. The article of claim 9 , wherein the device is a syringe.

11. The article of claim 10, wherein the syringe has an inner diameter of 1 to 5 mm.

12. A method for making the composition of claim 1, comprising: (a) producing decellularized omentum particles comprising pluripotent stem cells, the particles having a diameter of between 750 microns and 3 mm; (b) contacting the particles with at least one neuronal differentiation agent; (b) culturing the particles in the presence of the at least one neuronal differentiation agent under conditions that promote the generation of neural networks of mature neurons within the particles, thereby producing a composition according to any one of claims 1 to 7; A method comprising:

13. The method of claim 12, wherein the pluripotent stem cells are induced pluripotent stem cells, optionally reprogrammed from omental stromal cells.

14. 13. The method of claim 12, wherein the neuronal differentiation agent is selected from the group consisting of a transforming growth factor beta receptor 1 (ALK-5) inhibitor, a morphogenetic protein 4 (BMP4) inhibitor, retinoic acid, bone-derived neurotrophic factor (BDNF), ascorbic acid, and purmorphamine.

15. the mature neurons include motor neurons; and (i) greater than 50% of the motor neurons express neuron-specific class III β-tubulin (TUJ1) as measured by flow cytometry; and / or (ii) greater than 50% of the cells express motor neuron and pancreatic homeobox 1 (MNX1) as measured by flow cytometry; The method of claim 12.

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