Composition for cell transplantation therapy and use thereof

Abstract

Disclosed is a composition for supporting the survival and differentiation of neural progenitor cells (NPCs) transplanted into a site of nerve injury or disease, the composition comprising (a) a gel-forming molecule and (b) a chemokine receptor type 5 (CCR5) antagonist. Also disclosed is a method for treating nerve injury or disease in a subject, the method comprising: (a) mixing NPCs with a composition disclosed herein; and (b) administering the mixture of NPCs and the composition into the site of nerve injury or disease in the subject, thereby supporting the survival and differentiation of NPCs. Also disclosed is the use of a mixture of NPCs and a composition disclosed herein in the manufacture of a medicament for treating nerve injury or disease in a subject, the mixture being administered into the site of nerve injury or disease in the subject, thereby supporting the survival and differentiation of NPCs. Furthermore, disclosed is a kit for use in supporting the survival and differentiation of NPCs transplanted into the site of nerve injury or disease, the kit comprising: (a) a composition disclosed herein, (b) artificial cerebrospinal fluid (α-CSF), (c) CaCl2, and (d) thrombin.

Description

[Technical Field]

[0001] The present invention relates generally to compositions and uses thereof for supporting cell survival and differentiation in cell transplantation therapy, and in particular to compositions and uses thereof for supporting neural progenitor cell (NPC) survival and differentiation in NPC transplantation therapy for neural injury or disease. [Background technology]

[0002] Neurological injury or disease, including stroke, traumatic brain injury, and spinal cord injury, is a major cause of physical disability without effective treatment. For example, ischemic stroke results from a sudden decrease in cerebral blood flow and afflicts approximately 25% of people throughout their lives, accounting for nearly 5% of total disability-adjusted life years and 10% of total deaths worldwide. Conventional treatment is reperfusion in the acute phase of an ischemic event. However, there is no effective treatment beyond the acute phase. Therefore, there is an unmet need to develop a treatment for neurological injury or disease, such as ischemic stroke.

[0003] Stem cell-based approaches have shown promise as cell therapies, potentially protecting damaged neurons from further injury and / or replacing lost neurons. Transplantation of non-neuronal cells, such as mesenchymal stem cells (MSCs), has begun in clinical trials. MSCs have been shown to protect neurons in the penumbra, a brain region separating ischemic sites from healthy brain tissue, by regulating inflammation or angiogenesis. However, MSC transplantation does not result in neuronal replacement. Because the human brain has very limited regenerative capacity and MSCs do not normally generate neurons, it is desirable to have neural cells as a source for cell therapy.

[0004] Indeed, transplantation of neural progenitor cells (NPCs) in animal models of neurological conditions, such as stroke, spinal cord injury (SCI), and Parkinson's disease (PD), has demonstrated that these cells can mature into functional neurons and potentially integrate into the host brain circuitry. In animals with neuronal injury or disease, such as ischemic stroke, the site of neuronal injury or disease, e.g., the ischemic / infarct site, forms a cystic or cavitary lesion surrounded by a wall of glial scar and filled with inflammatory cells and secretions. This environment is unsuitable for transplanted NPCs, leading to poor survival of NPCs transplanted into the cystic or cavitary lesion and an inability for neural proliferation through the glial scar. Furthermore, the inhibitory environment promotes differentiation of transplanted NPCs into glial cells rather than neurons. Attempts to improve the survival of NPCs transplanted into the ischemic core have included high expression of small ubiquitin-like modifiers (SUMO), hypoxia treatment, co-transplantation with non-neuronal cells, and hydrogels crosslinked with biomaterials based on growth factors (e.g., bone morphogenetic protein 4 (BMP4), brain-derived neurotrophic factor (BDNF), and laminin-derived motifs (IKVAV)). In particular, hydrogels have anti-inflammatory properties and can be resorbed by tissue. Studies have demonstrated that hyaluronan-methylcellulose exhibits anti-inflammatory properties by reducing IL-1α levels in the central nervous system after stroke and spinal cord injury. Hydrogels could also be modified to modulate immune responses and promote angiogenesis, potentially enhancing the survival and differentiation of transplanted NPCs. When encapsulated in hydrogels, NPCs transplanted into the stroke cavity remained viable for two weeks with little proliferation. The anti-inflammatory polarizing effect of hydrogels on infiltrating microglia demonstrated the potential for inflammatory reprogramming of stroke lesions, which could contribute to neural regeneration after NPC transplantation. However, this method is unable to repopulate and reconstitute the injured brain due to the limited number of viable cells.The poor cell survival, even in the presence of neurotrophic support, compared to that in other neurological models, such as the 6-OHDA-induced Parkinson's model, led to the hypothesis underlying the present invention that modification of the unfavourable ischemic environment, particularly the inflammatory environment, is important for promoting NPC survival.

[0005] Currently, most studies transplant NPCs into the penumbra to avoid the hostile environment of cystic or cavitary lesions. Nevertheless, this creates additional damage to healthy brain regions. Furthermore, particularly when the ischemic cyst is relatively large, the cystic or cavitary lesion remains unfilled, leaving the separated brain regions unconnected. An alternative conventional method is to use a cocktail of growth factors to support the survival of NPCs transplanted into the injured spinal cord cyst. However, this requires the use of growth factors at 1000-fold physiological concentrations. This method actually requires large grafts to fill the cystic or cavitary lesion. However, this often results in the generation of tissue that occupies the spinal cord. Furthermore, this prevents neural progenitor cells from differentiating into mature neurons and glia, which are required for the transplanted cells to integrate into the host tissue and achieve therapeutic goals.

[0006] Transplantation of NPCs into cystic or cavitary lesions potentially fills this gap, replacing lost neurons and reconnecting disrupted circuits. Furthermore, efficient differentiation or maturation of transplanted NPCs is critical for cell transplantation therapy to function. Thus, there is a need for compositions and methods that enable and improve the survival of transplanted NPCs and promote the differentiation of transplanted NPCs into mature neurons, thereby reconstituting sites of neural injury or disease. Summary of the Invention [Means for solving the problem]

[0007] This disclosure describes a composition comprising two components: a gel-forming molecule and a chemokine receptor type 5 (CCR5) antagonist. In one example, the gel-forming molecule and CCR5 antagonist are FDA-approved drugs, fibrinogen and maraviroc, respectively. In the presence of the composition, NPCs transplanted into a site of neural injury or disease, such as the ischemic core, survive and differentiate into neurons, reconstituting the disrupted cortex.

[0008] In one aspect, the present disclosure provides a composition for supporting the survival and differentiation of neural progenitor cells (NPCs) transplanted into a site of neural injury or disease, comprising: (a) a gel-forming molecule, and (b) Chemokine receptor type 5 (CCR5) antagonist

[0023] The present invention refers to a composition comprising:

[0009] In another aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: (a) mixing NPC with a composition disclosed herein; (b) administering the mixture of NPCs and the composition into the subject at the site of neural injury or disease, thereby supporting the survival and differentiation of NPCs; The present invention refers to a method of treating a neurological injury or disease in a subject, comprising:

[0010] In another aspect, the present disclosure refers to the use of a mixture of NPCs and a composition disclosed herein in the manufacture of a medicament for treating neural injury or disease in a subject, wherein the mixture is administered into the site of neural injury or disease in the subject, thereby supporting the survival and differentiation of NPCs.

[0011] In another aspect, the present disclosure provides a kit for use in supporting the survival and differentiation of NPCs transplanted into a site of neural injury or disease, comprising: (a) a composition disclosed herein; (b) artificial cerebrospinal fluid (a-CSF); (c) CaCl2, and (d) thrombin This refers to a kit including:

[0012] Advantageously, the gel-forming molecules in the composition form a gel at 37°C after NPC transplantation, thereby acting as a scaffold to stabilize NPCs transplanted into cystic or cavitary lesions. The gel also prevents the CCR5 antagonist from being rapidly diluted. The CCR5 antagonist blocks signaling from inflammatory cytokines in cystic or cavitary lesions to CCR5 expressed on NPCs, reducing apoptosis of transplanted NPCs in inflammatory cystic or cavitary lesions and promoting differentiation of surviving NPCs. Additionally, the composition contains physiological concentrations of FDA-approved drugs, making it applicable in clinical settings.

[0013] The present invention is better understood by reference to the detailed description when considered in conjunction with the following non-limiting examples and the accompanying drawings. [Brief explanation of the drawings]

[0014] [Figure 1]Figure 1 illustrates the survival of NPCs transplanted into the ischemic core using the compositions disclosed herein. Figure 1a is a schematic diagram showing the process of NPC transplantation using fibrinogen and maraviroc. Figure 1b is a schematic diagram showing the experimental procedure and timeline for ischemic stroke induction, cell transplantation, and tissue collection for analysis. Figure 1c is a series of fluorescence microscopy images acquired using a Nikon Ti2 confocal microscope showing immunostaining for glial fibrillary acidic protein (GFAP) 7 days after transplantation in stroke mice transplanted with NPCs in artificial cerebrospinal fluid (a-CSF) alone, maraviroc alone, fibrinogen alone, or in the presence of the composition, showing GFP+ transplanted cells (green) within the cavitary lesion (outlined by a dashed line) and the GFAP+ glial scar surrounding the cavitary lesion. The dotted line outlines the ischemic core. The scale bar is 200 μm. Figure 1d is a series of fluorescence microscopy images of cortical slices acquired using a Nikon Ti2 confocal microscope showing cleaved caspase 3 (red) immunoreactivity in transplanted cells 7 days after transplantation. Separate fluorescence channels are shown below the microscope images. The scale bar is 100 μm. Figure 1e is a bar graph showing quantification of the percentage of cleaved caspase 3+ cells among GFP+ cells. n = 4 mice per group. Data are means ± SEM. Figure 1f is a series of fluorescence microscopy images acquired using a Nikon Ti2 confocal microscope illustrating DCX and SOX2 immunostaining in stroke mice transplanted with NPCs 7 days after transplantation. Yellow arrowheads indicate survival graphs. The dotted line outlines the ischemic core. The scale bar is 200 μm. Figure 1g is a dot plot illustrating the number of DCX+ or SOX2+ cells among GFP+ cells in various groups. n = 5 mice for the cocktail / composition group and n = 4 mice for all other three groups. Data are means ± SEM. [Figure 2]Figure 2 illustrates the maturation of NPCs transplanted into the ischemic core using the compositions disclosed herein. Figure 2a is a schematic diagram showing the procedure and timeline for ischemic stroke induction, cell transplantation, and tissue collection for analysis. Figure 2b is a fluorescent microscopy image acquired using a Nikon Ti2 confocal microscope showing the appearance of human cells (labeled with STEM121) transplanted into the cortical ischemic core (surrounded by a GFAP+ glial scar) 30 days post-transplant (dpt) in stroke mice transplanted with the cocktail / composition. Separate channels are shown on the right. The scale bar is 1 mm. LV is the lateral ventricle, and cc is the corpus callosum. Figure 2c is a whole-mount image of a brain from a mouse with (lower panel) or without (upper panel) NPC transplantation. The dotted line and black arrowhead indicate the site of injury or transplantation. The scale bar is 2 mm. Figure 2d shows serial coronal slices demonstrating complete filling of the stroke cavity with STEM121+ cells 30 days after transplantation (30 dpt). The scale bar is 1 mm. Figures 2e and 2f are fluorescent microscopy images acquired using a Nikon Ti2 confocal microscope showing neurofilament (NF) immunostaining, demonstrating the expression of NF in transplanted cells (GFP+ cells) at 30 dpt. The enlarged image in Figure 2f shows that GFP cells are NF-positive. The scale bar is 200 μm in Figure 2e and 100 μm in Figure 2f. Figure 2g is a pie chart showing quantification of the percentage of NF+ cells among GFP+ cells. n = 4 mice. Data are means ± SEM. Figure 2h is a fluorescent microscopy image acquired using a Nikon Ti2 confocal microscope showing immunostaining for STEM121 and NeuN, demonstrating the differentiation of transplanted cells into mature neurons 30 days after transplantation (30 dpt). The scale bar is 1 mm. LV is the lateral ventricle, and cc is the corpus callosum. Figure 2i is a bar graph showing the number of GFP+ NeuN+ or GFP+ NeuN- cells. n = 5 mice. Data are mean ± SEM. Figures 2j-2l are images of the area shown in Figure 2h, showing transplanted cells in the border (Figure 2j), upper layer (Figure 2k), and deep layer (Figure 2l). Scale bar is 100 μm. Figure 2m is a bar graph showing quantification of the percentage of NeuN+ cells in the upper and deep layer transplanted cells. n = 4 mice.Data are means ± SEM. p=0.0013, **p<0.01. [Figure 3a] Figure 3 illustrates glial reaction and angiogenesis in transplanted brains. Figure 3a is a series of fluorescence microscopy images acquired using a Nikon Ti2 confocal microscope showing immunostaining for Iba1 and GFAP in the brains of mice transplanted with NPCs in sham, a-CSF alone, maraviroc alone, fibrinogen alone, or the composition 30 days after transplantation. The scale bar is 200 μm. cc is the corpus callosum. Figure 3 illustrates glial reaction and angiogenesis in transplanted brains. Figure 3b is a magnified view showing the specific immunoreactivity of Iba1 and GFAP in variously treated mice. The asterisk indicates the ischemic core. The scale bar is 200 μm. Figure 3 illustrates glial reaction and angiogenesis in transplanted brains. Figure 3c is a series of fluorescence microscopy images acquired using a Nikon Ti2 confocal microscope showing immunostaining for CSPG and S100β, showing the glial reaction in mice treated with the composition compared to other groups. The asterisk indicates the ischemic core. The scale bar is 200 μm. [Figure 3b]Figure 3 illustrates glial reaction and angiogenesis in transplanted brains. Figure 3d is a bar graph showing quantification of indicated fluorescence intensity (normalized to the intact area) in the lesion site 30 days after transplantation. n=5 mice / group. Data are means ± SEM. **p=0.0017 in Figure 3d. Figure 3 illustrates glial reaction and angiogenesis in transplanted brains. Figure 3e is a bar graph showing quantification of indicated fluorescence intensity (normalized to the intact area) in the lesion site 30 days after transplantation. n=5 mice / group. Data are means ± SEM. *p=0.0372 in Figure 3e. Figure 3 illustrates glial reaction and angiogenesis in transplanted brains. Figure 3f is a bar graph showing quantification of indicated fluorescence intensity (normalized to the intact area) in the lesion site 30 days after transplantation. n=5 mice / group. Data are means ± SEM. ***p=0.0004 in Figure 3f. Figure 3 illustrates glial response and angiogenesis in transplanted brains. Figure 3g is a bar graph showing quantification of the amount of parenchyma within the damaged area surrounded by GFAP+ cells. n=5 mice / group. Data are means ± SEM. ***p=0.0009. Figure 3 illustrates glial response and angiogenesis in transplanted brains. Figure 3h is a series of fluorescent microscopy images acquired using a Nikon Ti2 confocal microscope showing immunostaining for STEM121 and laminin, demonstrating angiogenesis in the grafts 30 days after transplantation. The scale bar is 200 μm. Figure 3 illustrates glial response and angiogenesis in transplanted brains. Figure 3i is a bar graph showing quantification of blood vessel density in the graft and intact area. n=5 mice. Data are means ± SEM. ns p=0.1146. [Figure 4]Figure 4 illustrates the changes in expression of chemokine ligand (CCL) and chemokine receptor type 5 (CCR5) in transplanted brains. Figure 4a is a schematic diagram showing the experimental strategy shown in Figures 4b-4f. Figure 4b is a Western blot image showing the protein expression of CCR5, CCL3, CCL4, and CCL5 in the peri-infarct and infarct cortex at 2, 14, and 44 days post-stroke (dps). Figures 4c-4f are bar graphs showing quantification of protein expression of CCL3 (Figure 4c), CCL4 (Figure 4d), CCL5 (Figure 4e), and CCR5 (Figure 4f) normalized to GAPDH. n = 3 mice / group. Data are mean ± SEM. *p = 0.0136 in Figure 4c, *p = 0.0241 in Figure 4d, *p = 0.0499 in Figure 4e, and *p = 0.0244 in Figure 4f. Figure 4g is a schematic diagram illustrating the experimental strategy shown in Figures 4h-4l. Figure 4h is a Western blot image showing the protein expression of CCR5, CCL3, CCL4, and CCL5 in the graft and intact area 44 days after stroke. Figures 4i-4l are bar graphs showing the quantification of protein expression of CCL3 (Figure 4i), CCL4 (Figure 4j), CCL5 (Figure 4k), and CCR5 (Figure 4l) normalized to GAPDH. n = 3 mice / group in Figures 4i-4k, and n = 4 mice / group in Figure 4l. Data are means ± SEM. *p=0.0174 in Figure 4i, **p=0.0046 in Figure 4j, **p=0.0079 in Figure 4k, and **p=0.0066 in Figure 4l. [Figure 5]Figure 5 illustrates the expression and regulation of CCR5 in NPCs. Figures 5a-5c are fluorescence microscopy images acquired using a Nikon Ti2 confocal microscope showing immunostaining of CCR5 with SOX2 (Figure 5a), DCX (Figure 5b), and NeuN (Figure 5c), demonstrating CCR5 expression in NPCs, immature neurons, and mature neurons. Scale bars are 200 μm. Figure 5d shows a bar graph with Western blot images showing the expression levels of CCR5 on NPCs, immature neurons, and mature neurons. n = 3 samples per group. Data are means ± SEM and are compared with GAPDH. **p = 0.0094. Figure 5e is a schematic diagram illustrating the experimental strategy of Figures 5f-k. Figure 5f is a Western blot image showing the expression levels of CCR5 on NPCs in the presence or absence of CCL with or without CCR5-shRNA. Figures 5g-5h are fluorescence microscopy images acquired using a Nikon Ti2 confocal microscope. These are immunostaining images for SOX2 and CCR5, showing CCR5 expression on NPCs treated with vehicle (Fig. 5g) or CCR5-shRNA (Fig. 5h). Scale bar: 200 μm. Figure 5i is a Western blot image showing the expression level of CCR5 on the indicated cells. Figure 5j is a fluorescent microscopy image of NPCs obtained using a Nikon Ti2 confocal microscope, showing the number of apoptotic NPCs (TUNEL+) induced by CCL in the presence or absence of CCR5-shRNA. Scale bar: 200 μm. Figure 5k is a dot plot showing the quantification of the percentage of TUNEL+ cells in NPCs. n = 5 samples per group. Data are means ± SEM. ****p < 0.0001. Figure 5l is a schematic diagram showing that blocking CCR5 activation feedback attenuates NPC apoptosis. [Figure 6]Figure 6 illustrates the establishment of ischemic stroke by photothrombosis and the preparation of NPCs for transplantation. Figure 6a is a line graph showing the in vitro maraviroc release profile of the cocktail gel and free drug. Figure 6b is an image showing triphenyltetrazolium chloride (TTC) staining of brain slices, showing the infarct area 3 days post-stroke (dps). The black arrowhead indicates the infarct area. The scale bar is 1 mm. Figures 6c-6e are fluorescent microscopy images of NPCs acquired using a Nikon Ti2 confocal microscope, showing immunostaining for the cortical markers Brn2 (upper layer, c), Ctip2 (deep layer, d), and Foxp2 (deep layer, e), indicating various subtypes of cortical progenitor cells for transplantation. The scale bar is 200 μm. Figure 6f is a bar graph showing quantification of the percentage of indicated NPCs among GFP+ cells. n = 3 samples. Data are mean ± SEM. Figure 6g shows an image of dissociated GFP+ NPCs before transplantation. The scale bar is 200 μm. Figure 6h shows a fluorescence microscopy image of NPCs acquired using a Nikon Ti2 confocal microscope, showing immunostaining for the astrocyte markers GFAP and S100β, indicating that the ischemic core is surrounded by reactive astrocytes at 14 dps. The dotted lines indicate the ischemic core and corpus callosum (cc). The scale bar is 200 μm. Figure 6i shows a fluorescence microscopy image of NPCs acquired using a Nikon Ti2 confocal microscope, showing immunostaining for the neurite markers NF and microtubule-associated protein 2 (MAP2) on a cortical section, indicating that the injured cortex was collapsed and lacked neurites at 30 dps. The scale bar is 200 μm. [Figure 7]Figure 7 illustrates the survival and proliferation of transplanted NPCs. Figure 7a is a fluorescent microscopy image of NPCs acquired using a Nikon Ti2 confocal microscope showing immunostaining for NeuN. NPCs (GFP+) were transplanted into the ischemic core (NeuN-) 7 days after transplantation. The scale bar is 200 μm. PI is the infarct periphery, and cc is the corpus callosum. Figure 7b is a fluorescent microscopy image of NPCs acquired using a Nikon Ti2 confocal microscope showing immunostaining for Ki67. The expression of Ki67 in transplanted cells 7 days after transplantation is shown. The boxed area is enlarged in the lower panel. Yellow arrowheads indicate cells co-labeled with Ki67 and GFP. The scale bar is 200 μm. Figure 7c is a bar graph showing quantification of the percentage of Ki67+ cells among transplanted cells (GFP+). n = 4 mice per group. Data are mean ± SEM. [Figure 8] Figure 8 illustrates the survival of transplanted cells in the ischemic core at 30 days post-transplant. Figure 8a is a fluorescence microscopy image of NPCs acquired using a Nikon Ti2 confocal microscope showing immunostaining for NeuN and DCX using the indicated media in mice transplanted with NPCs (GFP+), demonstrating that GFP+ cells do not survive. The scale bar is 200 μm. Figure 8b is an image of a transplant from the cocktail / composition group showing Ki67 expression in transplanted cells (GFP+) 30 days post-transplant. The scale bar is 1 mm. Figure 8c is a pie chart showing quantification of the percentage of Ki67+ cells in transplanted cells. n=4 mice. Figure 8d is a fluorescence microscopy image of NPCs acquired using a Nikon Ti2 confocal microscope showing immunostaining for SOX9 30 days post-transplant in the cocktail / composition group of mice transplanted with NPCs. The scale bar is 1 mm. Figure 8e is a pie chart showing quantification of the percentage of SOX9+ cells in transplanted cells. n=4 mice. cc is the corpus callosum. Figure 8f shows GFP and laminin immunostaining showing the collapsed cortex in the four groups one month after transplantation. The scale bar is 500 μm. cc is the corpus callosum. [Figure 9]Figure 9 illustrates that cocktail-treated transplanted cells project axons from the ischemic core 30 days after transplantation. Figure 9a shows immunostaining for STEM121 and GFAP in a mouse transplanted with the cocktail and NPCs 30 days after transplantation, demonstrating axonal extension from the glial scar. Scale bar: 200 μm. Figure 9b shows immunostaining for STEM121 and the glutamatergic marker VGluT1, demonstrating differentiation of transplanted cells into glutamatergic neurons. Scale bar: 10 μm. Figure 9c shows immunostaining for STEM121, synapsin (a presynaptic marker), and psd95 (a postsynaptic marker) in the uninjured region adjacent to the injury site, indicating that neurites of transplanted neurons form synapses (white arrows) with host neurons. Scale bar: 20 μm. cc: corpus callosum. [Figure 10] Figure 10 illustrates the results of behavioral tests performed on days -14 (before stroke), 0, 14, and 30 after transplantation. Figure 10a shows quantification of fall latency in the rotarod test, indicating motor recovery 30 days after transplantation. n = 9 mice in the maraviroc and fibrinogen groups, n = 10 mice in all other groups. Data are means ± SEM. * p = 0.034 (cocktail vs. control). Figure 10b shows data on motor performance assessed by the grid-walk test. n = 9 mice in all groups in b. Data are means ± SEM. ns p = 0.053 (cocktail vs. control). [Figure 11a] Figure 11 illustrates that CCL induces apoptosis of NPCs. Figure 11a shows fluorescent microscopy images of NPCs acquired using a Nikon Ti2 confocal microscope showing immunostaining for SOX2, STEM121, and cleaved caspase-3, demonstrating the specific expression of cleaved caspase-3 in NPCs treated with the indicated treatments. The scale bar is 200 μm. [Figure 11b]Figure 11 illustrates that CCL induces apoptotic NPCs. Figure 11b is a bar graph showing quantification of the percentage of cleaved caspase 3+ cells in the presence of high (300 ng / mL) and low (10 ng / mL) concentrations of CCL. n = 5 samples in the low CCL group, n = 4 samples in the other four groups. Data are mean ± SEM. **p = 0.0058. Figure 11 illustrates that CCL induces apoptotic NPCs. Figure 11c is a phase-contrast image of live NPCs and immature neurons under the indicated treatments. The scale bar is 200 μm. [Figure 12] Figure 12 illustrates the effect of CCR5 inhibition on NPC survival. Figure 12a is a fluorescent microscopy image of NPC acquired using a Nikon Ti2 confocal microscope showing fluorescent staining of CCR5, STEM121, and TUNEL in NPCs with the indicated treatments. The scale bar is 200 μm. Figure 12b is a bar graph showing quantification of the percentage of apoptotic cells (TUNEL+) in NPCs in the presence of shRNA or maraviroc. n=3 samples / group. Data are mean±SEM. **p=0.0064, ****p<0.0001. [Figure 13] Figure 13 illustrates immunostaining of hNCAM (human neuronal marker) in the ischemic mouse brain. Projections of transplanted human neurons from the cortex to the brainstem are shown. The corresponding enlarged image is shown in the right panel. Scale bar: 1 mm. [Figure 14] Figure 14 illustrates immunostaining of hNCAM in the spinal cord of an ischemic mouse transplanted with human neurons. Axons of transplanted neurons are shown projecting into the spinal cord. Corresponding magnified images are shown in panels A and B. Scale bar: 200 μm. DETAILED DESCRIPTION OF THE INVENTION

[0015] This disclosure describes a composition containing an FDA-approved drug, e.g., fibrinogen and the CCR5 inhibitor maraviroc, that supports the survival and differentiation of NPCs transplanted into the brain at the site of neuronal injury or disease, thereby filling the brain's lesion gap, replacing lost cells, and reconnecting disrupted neural circuits, resulting in behavioral recovery in subjects suffering from neuronal injury or disease. This composition may be applied to cell transplantation therapy for neuronal injury or disease, such as stroke, traumatic brain injury, spinal cord injury, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and other neurological conditions characterized by inflammation.

[0016] In one aspect, the present disclosure refers to a composition for supporting the survival and differentiation of neural progenitor cells (NPCs) transplanted into a site of nerve injury or disease, the composition comprising: (a) a gel-forming molecule and (b) a chemokine receptor type 5 (CCR5) antagonist.

[0017] As used herein, the term "support" refers to maintaining, promoting, increasing or improving the proportion of NPCs that survive and differentiate into functional neurons and glial cells after transplantation into a site of nerve injury or disease.

[0018] As used herein, the term "survival" refers to the viability of a cell, in this case, an NPC, which is characterized by its ability to perform certain functions, such as metabolism, proliferation, regeneration, some forms of responsiveness, and adaptability. In one example, if an NPC transplanted into a site of nerve injury or disease does not express cleaved caspase 3, the NPC survives and is viable. In another example, if an NPC transplanted into a site of nerve injury or disease is negative for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, the NPC survives and is viable. In one example, at least 80% of the NPC survives in the presence of a composition disclosed herein after transplantation into a site of nerve injury or disease. In another example, at least 85% of the NPC survives in the presence of a composition disclosed herein after transplantation into a site of nerve injury or disease. In another example, at least 90% of the NPC survives in the presence of a composition disclosed herein after transplantation into a site of nerve injury or disease. In another example, at least 95% of NPCs are viable in the presence of a composition disclosed herein after transplantation into the site of nerve injury or disease. In another example, at least 99% of NPCs are viable in the presence of a composition disclosed herein after transplantation into the site of nerve injury or disease. In another example, about 100% of NPCs are viable in the presence of a composition disclosed herein after transplantation into the site of nerve injury or disease.

[0019] As used herein, the term "differentiation" refers to the transformation of NPCs into more specialized cell types, such as neurons and / or glial cells. In one example, NPCs differentiate into neurons in the presence of a composition disclosed herein after transplantation into a site of nerve injury or disease. In another example, the differentiated neuron is selected from the group consisting of bipolar neurons, multipolar neurons, and pseudounipolar neurons. In another example, the differentiated neuron is a bipolar neuron, having one axon and one dendrite extending from the cell body. In another example, the differentiated neuron is a multipolar neuron, having one axon and multiple dendrites. Multipolar neurons can be found in the central nervous system (CNS, consisting of the brain and spinal cord). In another example, a multipolar neuron is a Purkinje cell in the cerebellum, which has many branched dendrites but only one axon. In another example, the differentiated neuron is a pseudounipolar neuron, having a single process extending from the cell body, which later branches into two separate structures like a bipolar cell. In one example, pseudounipolar neurons are sensory neurons with an axon that branches into two extensions, one of which connects to a dendrite that receives sensory information and the other of which transmits this information to the spinal cord. In another example, NPCs, after transplantation into the site of nerve injury or disease, differentiate into glial cells in the presence of a composition disclosed herein. In another example, the differentiated glial cells are selected from the group consisting of astrocytes, microglia, oligodendrocytes, radial glia, and ependymal cells. In another example, the differentiated glial cells are astrocytes, which contact both capillaries and neurons in the CNS to supply nutrients and other substances to neurons, regulate the concentrations of ions and chemicals in the extracellular fluid, and provide structural support for synapses. In another example, the differentiated glial cells are microglia, which remove and degrade dead cells and protect the brain from invading microorganisms. In another example, the differentiated glial cells are oligodendrocytes, which form myelin sheaths around axons in the CNS. In another example, the differentiated glial cells are radial glia, which act as a scaffold for developing neurons as they migrate to their final destination. In another example, the differentiated glial cells are ependymal cells, which line the fluid-filled ventricles of the brain and the central canal of the spinal cord.

[0020] In one example, NPCs differentiate into neurons in the presence of a composition disclosed herein after transplantation into a site of nerve injury or disease. In one example, 85% to 95% of NPCs differentiate into neurons in the presence of a composition disclosed herein after transplantation into a site of nerve injury or disease. In another example, 85% to 90% of NPCs differentiate into neurons in the presence of a composition disclosed herein after transplantation into a site of nerve injury or disease. In another example, 90% to 95% of NPCs differentiate into neurons in the presence of a composition disclosed herein after transplantation into a site of nerve injury or disease. In another example, about 85% of NPCs differentiate into neurons in the presence of a composition disclosed herein after transplantation into a site of nerve injury or disease. In another example, about 86% of NPCs differentiate into neurons in the presence of a composition disclosed herein after transplantation into a site of nerve injury or disease. In another example, about 87% of NPCs differentiate into neurons in the presence of a composition disclosed herein after transplantation into a site of nerve injury or disease. In another example, after transplantation into the site of nerve injury or disease, in the presence of a composition disclosed herein, about 88% of NPCs differentiate into neurons. In another example, after transplantation into the site of nerve injury or disease, in the presence of a composition disclosed herein, about 89% of NPCs differentiate into neurons. In another example, after transplantation into the site of nerve injury or disease, in the presence of a composition disclosed herein, about 90% of NPCs differentiate into neurons. In another example, after transplantation into the site of nerve injury or disease, in the presence of a composition disclosed herein, about 91% of NPCs differentiate into neurons. In another example, after transplantation into the site of nerve injury or disease, in the presence of a composition disclosed herein, about 92% of NPCs differentiate into neurons. In another example, after transplantation into the site of nerve injury or disease, in the presence of a composition disclosed herein, about 93% of NPCs differentiate into neurons. In another example, after transplantation into the site of nerve injury or disease, in the presence of a composition disclosed herein, about 94% of NPCs differentiate into neurons. In another example, after transplantation into the site of nerve injury or disease, in the presence of a composition disclosed herein, about 95% of NPCs differentiate into neurons. In another example, after transplantation into the site of nerve injury or disease, in the presence of a composition disclosed herein, 5% to 15% of NPCs differentiate into glial cells.In another example, after transplantation into a site of nerve injury or disease, in the presence of a composition disclosed herein, 5% to 10% of NPCs differentiate into glial cells. In another example, after transplantation into a site of nerve injury or disease, in the presence of a composition disclosed herein, 10% to 15% of NPCs differentiate into glial cells. In another example, after transplantation into a site of nerve injury or disease, in the presence of a composition disclosed herein, about 5% of NPCs differentiate into glial cells. In another example, after transplantation into a site of nerve injury or disease, in the presence of a composition disclosed herein, about 10% of NPCs differentiate into glial cells. In another example, after transplantation into a site of nerve injury or disease, in the presence of a composition disclosed herein, about 15% of NPCs differentiate into glial cells.

[0021] In one example, NPCs differentiate into neurons and / or glial cells in the presence of a composition disclosed herein about 1 to 6 months after transplantation into the site of nerve injury or disease. In another example, NPCs differentiate into neurons and / or glial cells in the presence of a composition disclosed herein about 30 days after transplantation into the site of nerve injury or disease. In another example, NPCs differentiate into neurons and / or glial cells in the presence of a composition disclosed herein about 40 days after transplantation into the site of nerve injury or disease. In another example, NPCs differentiate into neurons and / or glial cells in the presence of a composition disclosed herein about 50 days after transplantation into the site of nerve injury or disease. In another example, NPCs differentiate into neurons and / or glial cells in the presence of a composition disclosed herein about 60 days after transplantation into the site of nerve injury or disease. In another example, NPCs differentiate into neurons and / or glial cells in the presence of a composition disclosed herein about 3 months after transplantation into the site of nerve injury or disease. In another example, NPCs differentiate into neurons and / or glial cells in the presence of a composition disclosed herein about four months after transplantation into the site of nerve injury or disease. In another example, NPCs differentiate into neurons and / or glial cells in the presence of a composition disclosed herein about five months after transplantation into the site of nerve injury or disease. In another example, NPCs differentiate into neurons and / or glial cells in the presence of a composition disclosed herein about six months after transplantation into the site of nerve injury or disease. In one example, the neurons into which NPCs differentiate project axons to CNS tissues, e.g., the brainstem, spinal cord, and contralateral cortex, reconnecting the ischemic cortex with the rest of the brain about six months after transplantation in the presence of a composition disclosed herein.

[0022] The survival and differentiation of NPCs transplanted into sites of nerve injury or disease are supported by the compositions disclosed herein, comprising a gel-forming molecule and a CCR5 antagonist. In one example, the gel-forming molecule is fibrinogen. The fibrinogen molecule is a 340 kDa homodimeric glycoprotein composed of two Aα, two Bβ, and two γ polypeptide chains linked by 29 disulfide bridges. In the presence of thrombin, thrombin cleaves fibrinopeptides to form fibrin monomers. These monomers then polymerize into semi-staggered arrays to form fibrin protofibrils, ultimately forming a fibrin network, an insoluble gel. Furthermore, calcium significantly accelerates the fibrin monomer polymerization step, thereby shortening the time required for fibrinogen to form fibrin. Advantageously, when fibrinogen forms a gel in the presence of thrombin or calcium, the gel provides a scaffold that stabilizes NPCs transplanted into sites of nerve injury or disease. Additionally, fibrinogen is neurotrophic and supports the growth of transplanted NPCs. Furthermore, the gel-formed fibrinogen prevents rapid dilution and / or degradation of maraviroc, supporting the survival of transplanted NPCs.

[0023] In one example, the fibrinogen in the compositions disclosed herein is at a concentration of 5 mg / mL to 30 mg / mL. In another example, the fibrinogen in the compositions disclosed herein is at a concentration of about 5 mg / mL. In another example, the fibrinogen in the compositions disclosed herein is at a concentration of about 9 mg / mL. In another example, the fibrinogen in the compositions disclosed herein is at a concentration of about 10 mg / mL. In another example, the fibrinogen in the compositions disclosed herein is at a concentration of about 15 mg / mL. In another example, the fibrinogen in the compositions disclosed herein is at a concentration of about 20 mg / mL. In another example, the fibrinogen in the compositions disclosed herein is at a concentration of about 25 mg / mL. In another example, the fibrinogen in the compositions disclosed herein is at a concentration of about 30 mg / mL.

[0024] In another example, the gel-forming molecule is agarose. In another example, the gel-forming molecule is collagen. In another example, the gel-forming molecule is gelatin. In another example, the gel-forming molecule is chitosan. In another example, the gel-forming molecule is alginate. In another example, the gel-forming molecule is fibrin. In another example, the gel-forming molecule is hyaluronic acid. In another example, the gel-forming molecule is laminin. In another example, the gel-forming molecule is a degradable polymer selected from the group consisting of poly(glycolic acid) (PGA), poly(caprolactone) (PCL), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), and poly(ethylene glycol) (PEG).

[0025] The compositions disclosed herein include CCR5 antagonists. CCR5 is an inflammatory chemokine receptor. In the immune system, CCR5 is predominantly expressed on T cells, macrophages, dendritic cells, and eosinophils. Effector CCR5 + T cells are directed to sites of infection and inflammation by chemokines produced in local tissues, activating innate immune cells at the site and triggering a cascade of innate immune responses. CCR5 is one of the receptors for chemokine ligand 3 (CCL3), CCL4, and CCL5. CCL3 and CCL4 are two protein components of macrophage inflammatory protein (MIP) 1, also named MIP1 alpha and MIP1 beta, respectively. CCR5 is expressed on neural stem cells (NSCs) or neural progenitor cells, but not on mature neurons. This is because, as a chemokine receptor, in the case of nerve injury or disease, CCR5 is directed to the site of nerve injury or disease, which has a CCR5 ligand (e.g., a chemokine resulting from inflammation). + It helps in cell recruitment. Thus, NSCs or neural progenitor cells tend to form clusters or neural rosettes at the site of nerve injury or disease, which maintains the NSCs or neural progenitor cells in an immature stem or progenitor cell state. When CCR5 signaling is blocked, the cluster formation becomes less or the distribution becomes more uniform as the cells differentiate or mature.

[0026] As used herein, the term "CCR5 antagonist" refers to a molecule that inhibits, attenuates, or reduces the biological activity of CCR5 or reduces the protein level of CCR5. In one example, a CCR5 antagonist is a molecule that inhibits, attenuates, or reduces the biological activity of CCR5 by interfering with the interaction of CCR5 with another molecule, for example, its ligand CCL3 / 4 / 5. In one example, a CCR5 antagonist is a molecule that inhibits, attenuates, or reduces the biological activity of CCR5 by acting on a component of a biological pathway in which CCR5 is involved. In another example, a CCR5 antagonist is a molecule that reduces the protein level of CCR5 by reducing the expression of the gene encoding CCR5. The CCR5 antagonist may be a molecule selected from the group consisting of a small molecule, a nucleic acid, an antibody, an anticalin, a carbohydrate, and any other compound or composition that inhibits, attenuates or reduces the activity of CCR5 by directly interacting with CCR5, or by acting on a component of a biological pathway in which CCR5 is involved, or by reducing the protein expression level of CCR5.

[0027] In one example, the CCR5 antagonist is a small molecule. In another example, the CCR5 antagonist is maraviroc. Maraviroc (trade name Selzentry or Celsentri outside the United States) is a chemokine receptor antagonist developed by the pharmaceutical company Pfizer that acts against HIV by interfering with the interaction between HIV and CCR5. It was approved for use by the FDA in August 2007. In one example, maraviroc blocks the activation of CCR5 expressed on transplanted NPCs by inflammatory cytokines such as CCL released by inflammatory cells infiltrating the site of neural injury or disease, thereby reducing apoptosis and promoting the differentiation / maturation of the transplanted NPCs. Thus, maraviroc can effectively reduce inflammatory injury in the inappropriate inflammatory environment of the site of neural injury or disease, such as the infarct core.

[0028] In one example, the maraviroc in the compositions disclosed herein is at a concentration of 3 mg / mL to 50 mg / mL. In another example, the maraviroc in the compositions disclosed herein is at a concentration of about 3 mg / mL. In another example, the maraviroc in the compositions disclosed herein is at a concentration of about 5 mg / mL. In another example, the maraviroc in the compositions disclosed herein is at a concentration of about 10 mg / mL. In another example, the maraviroc in the compositions disclosed herein is at a concentration of about 15 mg / mL. In another example, the maraviroc in the compositions disclosed herein is at a concentration of about 20 mg / mL. In another example, the maraviroc in the compositions disclosed herein is at a concentration of about 25 mg / mL. In another example, the maraviroc in the compositions disclosed herein is at a concentration of about 30 mg / mL. In another example, the maraviroc in the compositions disclosed herein is at a concentration of about 35 mg / mL. In another example, the maraviroc in the compositions disclosed herein is at a concentration of about 40 mg / mL. In another example, the maraviroc in the compositions disclosed herein is at a concentration of about 45 mg / mL. In another example, the maraviroc in the compositions disclosed herein is at a concentration of about 50 mg / mL.

[0029] In another example, the CCR5 antagonist is a small molecule selected from the group consisting of Fuscin, TAK-220, Nifeviroc, DAPTA, Aplaviroc, Aplaviroc Hydrochloride, Ophiobolin C, AZD-5672, and Maraviroc-d6.

[0030] In another example, the CCR5 antagonist is a nucleic acid. In another example, the CCR5 antagonist is a small interfering RNA (siRNA). Small interfering RNA (siRNA) is typically a double-stranded RNA molecule 20 to 25 nucleotides in length. When transfected into cells, siRNA transiently inhibits target mRNA until it is degraded within the cell. In another example, the CCR5 antagonist is a small hairpin RNA (shRNA). Small hairpin RNA (shRNA) is typically an RNA sequence approximately 80 base pairs in length, containing an internal hybridization region that generates a hairpin structure. The shRNA molecule is processed within the cell to form siRNA, which then knocks down gene expression. In another example, the CCR5 antagonist is a microRNA (miRNA). miRNA is a small, non-coding RNA with an average length of 22 nucleotides. miRNAs are partially complementary to one or more messenger RNA (mRNA) molecules and can downregulate gene expression in a variety of ways, including translational repression, mRNA cleavage, and deadenylation.

[0031] In one example, the siRNA in the compositions disclosed herein is at least 1×10 6 ~1×10 8 In another example, the shRNA in the compositions disclosed herein is at a concentration of 1 x 10 U / mL. 6 ~1×10 8 In another example, the miRNA in the compositions disclosed herein is at a concentration of 1 x 10 U / mL. 6 ~1×10 8 In another example, the siRNA or shRNA or miRNA in the compositions disclosed herein is at a concentration of about 1 x 10 6 In another example, the siRNA or shRNA or miRNA in the compositions disclosed herein is at a concentration of about 5×10 6 In another example, the siRNA or shRNA or miRNA in the compositions disclosed herein is at a concentration of about 1 x 10 7In another example, the siRNA or shRNA or miRNA in the compositions disclosed herein is at a concentration of about 5×10 7 In another example, the siRNA or shRNA or miRNA in the compositions disclosed herein is at a concentration of about 1 x 10 8 The concentration is in U / mL.

[0032] In one example, the nucleic acid-based CCR5 antagonist is delivered to NPCs using a viral vector. In another example, the viral vector is a lentiviral vector. In another example, the viral vector is an adenoviral vector. In another example, the viral vector is an adeno-associated virus (AAV) vector. In another example, the viral vector is a retroviral vector. Advantageously, viral vectors usually result in high transfection efficiency. In another example, the nucleic acid-based CCR5 antagonist is delivered to NPCs using a non-viral vector. In one example, the non-viral vector is a vector based on an inorganic material selected from the group consisting of gold nanoparticles (AuNPs), mesoporous silicon, graphene oxide, and Fe3O4-mediated nanoparticles (NPs). In another example, the non-viral vector is a nanocarrier based on a cationic lipid, such as lipofectamine, and a neutral lipid, such as cholesterol, dioleoylphosphatidylcholine (DOPC), and dioleoylphosphatidylethanolamine (DOPE). In another example, the non-viral vector is a polymer vector selected from the group consisting of polyethyleneimine (PEI), polylactic-co-glycolic acid (PLGA), chitosan, and β-cyclodextrin, hi another example, the non-viral vector is a dendrimer-based vector, e.g., a PAMAM dendrimer-based vector.

[0033] In another example, the CCR5 antagonist is an antibody. In another example, the CCR5 antagonist is the PRO 140 antibody (Leronlimab).

[0034] As used herein, the term "neural progenitor cells (NPCs)" refers to a mixed population of cells consisting of all undifferentiated progeny of neural stem cells (NSCs), and thus includes both NSCs and neural progenitor cells. The term "neural progenitor cells (NPCs)" is generally used to collectively describe a mixed population of NSCs and neural progenitor cells.

[0035] As used herein, the term "neural stem cell (NSC)" refers to a multipotent cell of the central nervous system (CNS, comprised of the brain and spinal cord) that is capable of self-renewal and proliferation without restriction, and of generating progeny that terminally differentiate into many, but not all, of the glial and neural cell types that colonize the CNS, e.g., neurons, or glial cells, including astrocytes and oligodendrocytes. Non-stem cell NSC progeny are also referred to as neural progenitor cells.

[0036] As used herein, the term "neural progenitor cell" refers to a cell that has the ability to proliferate and differentiate into at least one cell type. Thus, neural progenitor cells can be unipotent, bipotent, or multipotent. A distinctive feature of neural progenitor cells is that, unlike stem cells, they have limited proliferation capacity and do not exhibit self-renewal.

[0037] In one example, NPCs are generated in vitro by differentiating embryonic stem cells (ESCs). In another example, NPCs are generated in vitro by differentiating induced pluripotent stem cells (iPSCs). iPSCs are derived from adult cells, most often fibroblasts or blood cells, and programmed to an embryonic-like pluripotent state.

[0038] In one example, the NPCs are embryonic NPCs isolated from the CNS of a developing embryo. During mammalian CNS development, NPCs arising from the neural tube generate a pool of multipotent neural progenitor cells and more restricted neural progenitor cells, which then proliferate, migrate, and further differentiate into neurons and glial cells. During embryogenesis, NPCs are derived from the neuroectoderm and can first be detected in the formation of the neural plate and neural tube. As the embryo develops, NSCs can be identified in almost all regions of the embryonic CNS, including the septum, cortex, thalamus, ventral midbrain, and spinal cord. NSCs isolated from these regions have distinct spatial identities and differentiation potentials.

[0039] In another example, the NPCs are adult NPCs isolated from the CNS of a mature adult. In another example, the adult NPCs are found in a region of the CNS of a mature adult selected from the group consisting of the subgranular zone within the dentate gyrus of the hippocampus, the subventricular zone around the lateral ventricles, and the hypothalamus (specifically within the dorsal alpha 1 region, alpha 2 region, and "hypothalamic proliferative zone," located in the adjacent median eminence).

[0040] NPCs may be transplanted into a site of neural injury or disease together with a composition disclosed herein that supports the survival and differentiation of the transplanted NPCs. As used herein, the term "neural injury or disease site" refers to a site resulting from neural injury or disease characterized by inflammation and selected from the group consisting of stroke, traumatic brain injury, spinal cord injury, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, hypoxia-induced neuronal cell injury such as that seen in cardiac arrest or neonatal distress, neurological conditions associated with cancer, and neurodegenerative diseases. In one example, the neural injury or disease site results from a stroke, particularly a major stroke. A major stroke is the sudden and permanent death of brain cells that occurs when blood flow is blocked, preventing oxygen from being delivered to the brain. In one example, the major stroke is an ischemic stroke. Ischemic stroke most commonly occurs when blood flow is obstructed by a clot (known as "thrombosis" of the artery) or a dislodged blood clot lodged within the artery (also called an "embolic stroke"). In another example, a major stroke is a hemorrhagic stroke. Hemorrhagic stroke results from a rupture of the arterial wall and leakage of blood into the surrounding brain. Hemorrhagic stroke, like ischemic stroke, causes tissue death by depriving the brain of blood and oxygen, resulting in many neurological disorders (movement, speech) as well as functional impairment. In another example, nerve damage or disease results from a traumatic brain injury. Traumatic brain injury usually results from a severe blow or impact to the head or body. Traumatic brain injury can also be caused by an object passing through brain tissue, such as a bullet or shattered skull fragments. Traumatic brain injury can result in subcutaneous bleeding, tissue tearing, bleeding, and other physical damage to the brain. Such damage can lead to long-term complications as well as death. In one example, the traumatic brain injury is a closed brain injury. A closed brain injury occurs when a non-penetrating injury to the brain occurs due to rapid back and forth movement and concussion of the brain inside the skull without disrupting the skull, resulting in subcutaneous bleeding and rupture of brain tissue and blood vessels. In another example, the traumatic brain injury is a penetrating brain injury. A penetrating or open head injury occurs when there is disruption of the skull, for example, when a bullet penetrates the brain. In another example, the nerve damage or disease site results from a spinal cord injury.Spinal cord injury refers to damage to any part of the spinal cord or nerves at the end of the spinal canal (cauda equina), which often results in permanent changes in strength, sensation, and other bodily functions below the injury site. In another example, nerve damage or diseased areas result from multiple sclerosis (MS). In MS, the immune system attacks the protective sheath (myelin) that covers nerve fibers, which can cause permanent damage or deterioration of the nerves. In another example, nerve damage or diseased areas result from Alzheimer's disease, which is characterized by nerve cell damage. In another example, nerve damage or diseased areas result from Parkinson's disease. In Parkinson's disease, nerve cells in the basal ganglia, the region of the brain that controls movement, are damaged and / or die. In another example, nerve damage or diseased areas result from Huntington's disease, which results in movement disorders, cognitive impairment, and psychiatric disorders with a wide range of signs and symptoms. In another example, nerve damage or diseased areas result from amyotrophic lateral sclerosis (ALS). ALS affects nerve cells (motor neurons) that control voluntary muscle movements, such as walking and speaking, leading to the gradual deterioration and subsequent death of motor neurons.

[0041] In one example, the nerve damage or disease site is in the brain. In another example, the nerve damage or disease site is in the spinal cord. In another example, the nerve damage or disease site is in the cerebrum. In another example, the nerve damage or disease site is in the cerebellum. In another example, the nerve damage or disease site is in the brainstem. In another example, the nerve damage or disease site is in the frontal lobe of the cerebrum. In another example, the nerve damage or disease site is in the parietal lobe of the cerebrum. In another example, the nerve damage or disease site is in the occipital lobe of the cerebrum. In another example, the nerve damage or disease site is in the temporal lobe of the cerebrum. In another example, the nerve damage or disease site is in the midbrain of the brainstem. In another example, the nerve damage or disease site is in the pons of the brainstem. In another example, the nerve damage or disease site is in the medulla. In another example, the nerve damage or disease site is in two or more of the above sites. In another example, the nerve damage or disease site is located in a brain region selected from the group consisting of the pituitary gland, hypothalamus, amygdala, hippocampus, pineal gland, ventricles, and cerebrospinal fluid. In another example, the nerve damage or disease site comprises injury to a cranial nerve selected from the group consisting of the olfactory nerve, optic nerve, oculomotor nerve, trochlear nerve, trigeminal nerve, abducens nerve, facial nerve, vestibulocochlear nerve, glossopharyngeal nerve, vagus nerve, accessory nerve, and hypoglossal nerve. In another example, the nerve damage or disease site comprises injury to a blood vessel selected from the group consisting of the basilar artery, vertebral artery, external carotid artery, internal carotid artery, and circle of Willis. In another example, the nerve damage or disease site is focal (limited to one brain region). In another example, the nerve damage or disease site is diffuse (occurring in two or more brain regions).

[0042] In animals with a neuronal injury or disease disclosed herein, such as an ischemic stroke, the site of neuronal injury or disease has neuronal cell death or vascular injury, or both. In one example, the ischemic / infarct site is surrounded by a wall of glial scarring, forming a cavity filled with inflammatory cells and secretions. This inflammatory environment is unsuitable for transplanted NPCs, rendering most transplanted NPCs nonviable. The compositions disclosed herein support the survival and differentiation of NPCs transplanted into the site of a neuronal injury or disease disclosed herein.

[0043] In one example, a composition comprising a gel-forming molecule and a CCR5 antagonist disclosed herein further comprises calcium. In another example, a composition comprising fibrin and maraviroc further comprises CaCl2. Advantageously, calcium significantly accelerates the steps of fibrin monomer polymerization and gel formation, thereby shortening the time required for fibrinogen to form fibrin. The gel efficiently holds together the transplanted NPCs and prevents rapid dilution and / or degradation of maraviroc, supporting the survival of the transplanted NPCs. In one example, the CaCl2 in the composition disclosed herein is at a concentration of 1-5 mM. In a specific example, the CaCl2 in the composition disclosed herein is at a concentration of 2.5 mM.

[0044] In another example, the composition comprising the gel-forming molecule and the CCR5 antagonist disclosed herein does not contain calcium. After administration of a mixture of NPC and the composition disclosed herein to the site of nerve injury or disease, calcium present in bodily fluids promotes gel formation.

[0045] In another example, a composition comprising a gel-forming molecule and a CCR5 antagonist disclosed herein further comprises thrombin. Thrombin mediates proteolytic cleavage and removal of N-terminal fibrinopeptides from the Aα and Bβ chains of fibrinogen, resulting in fibrin (gel) formation. In one example, the thrombin in the composition disclosed herein is at a concentration of 10 U / mL to 500 U / mL. In another example, the thrombin in the composition disclosed herein is at a concentration of about 10 U / mL. In another example, the thrombin in the composition disclosed herein is at a concentration of about 50 U / mL. In another example, the thrombin in the composition disclosed herein is at a concentration of about 100 U / mL. In another example, the thrombin in the composition disclosed herein is at a concentration of about 200 U / mL. In another example, the thrombin in the composition disclosed herein is at a concentration of about 300 U / mL. In another example, the thrombin in the composition disclosed herein is at a concentration of about 400 U / mL. In another example, the thrombin in the compositions disclosed herein is at a concentration of about 500 U / mL.

[0046] In another example, the composition comprising the gel-forming molecule and the CCR5 antagonist disclosed herein does not contain thrombin. After administration of a mixture of NPC and the composition disclosed herein to the site of nerve injury or disease, thrombin present in bodily fluids promotes gel formation.

[0047] In another example, the compositions disclosed herein further comprise a growth factor selected from the group consisting of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin, platelet-derived growth factor (PDGF), glial cell line-derived neurotrophic factor (GDNF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and bone morphogenetic protein 4 (BMP4). The growth factor may be used in the compositions disclosed herein depending on the nature of the NPCs, the number of NPCs to be transplanted, and the size of the lesion to be filled. In one example, the growth factor disclosed herein is included in the composition when the site of nerve injury or disease, for example, the lesion is large and requires additional cell division to fill it. In another example, the brain-derived neurotrophic factor (BDNF) in the composition is present at a concentration of 1 ng / mL to 100 ng / mL. In another example, the nerve growth factor (NGF) in the composition is at a concentration of 1 ng / mL to 100 ng / mL. In another example, the neurotrophin in the composition is at a concentration of 1 ng / mL to 50 ng / mL. In another example, the platelet-derived growth factor (PDGF) in the composition is at a concentration of 1 ng / mL to 100 ng / mL. In another example, the glial cell line-derived neurotrophic factor (GDNF) in the composition is at a concentration of 1 ng / mL to 500 ng / mL. In another example, the insulin-like growth factor 1 (IGF-1) in the composition is at a concentration of 1 ng / mL to 50 ng / mL. In another example, the insulin-like growth factor 2 (IGF-2) in the composition is at a concentration of 1 ng / mL to 50 ng / mL. In another example, the fibroblast growth factor (FGF) in the composition is at a concentration of 1 ng / mL to 200 ng / mL. In another example, the vascular endothelial growth factor (VEGF) in the composition is at a concentration of 10 ng / mL to 500 ng / mL. In another example, the bone morphogenetic protein (BMP4) in the composition is present at a concentration of 5 ng / mL to 500 ng / mL. Advantageously, the concentrations of growth factors in the compositions disclosed herein are comparable to these physiological concentrations. This avoids the problem of conventional use in the art of microgram levels of growth factors in conjunction with fibrinogen to support cells transplanted into the injury site, i.e., the conventionally used growth factor concentrations are more than 1000 times the physiological concentration.At such high concentrations, such growth factors promote the proliferation of transplanted NPCs, forming "space-occupying" tissues, e.g., tumors, within the spinal cord, which can cause secondary damage by compressing the intact area. Furthermore, the use of high concentrations (more than 1000 times the physiological concentration) of growth factors prevents the differentiation of NPCs. Thus, in previous studies, transplanted NPCs maintained a neural progenitor state for many months. In contrast, the concentrations of growth factors used herein maintain transplanted NPCs in a proliferative state, avoiding the scenario of overgrowth that leads to "space-occupying" tissues.

[0048] In another example, the compositions disclosed herein do not contain a growth factor selected from the group consisting of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), glial cell line-derived neurotrophic factor (GDNF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and bone morphogenetic protein 4 (BMP4), which prevents overgrowth of transplanted NPCs in the presence of relatively small lesions.

[0049] In another aspect, the present disclosure refers to a method of treating neural injury or disease in a subject, comprising: (a) combining NPCs with a composition disclosed herein; and (b) administering the combination of NPCs and the composition into the site of the neural injury or disease in the subject, thereby supporting survival and differentiation of the NPCs.

[0050] In another aspect, the present disclosure refers to the use of a mixture of NPCs and a composition disclosed herein in the manufacture of a medicament for treating neural injury or disease in a subject, wherein the mixture is administered into the site of neural injury or disease in the subject, thereby supporting the survival and differentiation of NPCs.

[0051] As used herein, the term "treatment" and grammatical variants of this term refer to the administration of a mixture of NPC and a composition disclosed herein to a subject as described herein by any suitable means as described herein. Such treatment includes any and all uses that treat a disease state or symptom, prevent the establishment of a disease, or otherwise prevent, hinder, slow, or reverse the progression of a disease or other undesirable symptom in any and all ways.

[0052] As disclosed herein, the term "neural progenitor cells (NPCs)" is used to collectively describe a mixed population of NSCs and neural precursor cells. In one example, NPCs are generated in vitro by differentiating embryonic stem cells (ESCs). In another example, NPCs are generated in vitro by differentiating induced pluripotent stem cells (iPSCs). iPSCs are derived from adult cells, most often fibroblasts or blood cells, and programmed to an embryonic-like pluripotent state. In one example, NPCs are embryonic NPCs isolated from the CNS of a developing embryo. During mammalian CNS development, NPCs arising from the neural tube generate pools of multipotent neural precursor cells and more restricted neural progenitor cells, which then proliferate, migrate, and further differentiate into neurons and glial cells. During embryogenesis, NPCs are derived from the neuroectoderm and can first be detected in the forming neural plate and neural tube. As the embryo develops, NSCs can be identified in almost all regions of the embryonic CNS, including the septum, cortex, thalamus, ventral midbrain, and spinal cord. NSCs isolated from these regions have distinct spatial identities and differentiation potential. In another example, the NPCs are adult NPCs isolated from the CNS of a mature adult. In another example, adult NPCs are found in regions of the CNS of a mature adult selected from the group consisting of the subgranular zone in the dentate gyrus of the hippocampus, the subventricular zone surrounding the lateral ventricles, and the hypothalamus (specifically, in the dorsal α1 region, α2 region, and "hypothalamic proliferative zone," located adjacent to the median eminence).

[0053] In one example, the neurological injury or disease disclosed herein is a disease or disorder of the central nervous system, including, but not limited to, stroke, traumatic brain injury, spinal cord injury, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, hypoxia-induced neuronal cell injury such as seen in cardiac arrest or neonatal distress, neurological conditions associated with cancer, and neurodegenerative diseases. In one example, the neurological injury or disease is a stroke, particularly a cerebral stroke. A cerebral stroke is the sudden and permanent death of brain cells that occurs when blood flow is blocked, preventing oxygen from being delivered to the brain. In one example, the cerebral stroke is an ischemic stroke. An ischemic stroke most commonly occurs when blood flow is obstructed by clotting (known as arterial "thrombosis") or by the detachment of a blood clot lodged in an artery (also called an "embolic stroke"). In another example, the stroke is a hemorrhagic stroke. Hemorrhagic stroke results from the rupture of an arterial wall and the leakage of blood into the surrounding brain. Hemorrhagic stroke, like ischemic stroke, causes tissue death by depriving the brain of blood and oxygen, resulting in many neurological disorders (movement, speech) as well as functional impairments. In another example, the neurological damage or disease is a traumatic brain injury. Traumatic brain injury usually results from a severe blow or impact to the head or body. Traumatic brain injury can also be caused by an object passing through brain tissue, such as a bullet or shattered skull fragments. Traumatic brain injury can result in subcutaneous bleeding, tissue tearing, bleeding, and other physical damage to the brain. Such injuries can lead to long-term complications as well as death. In one example, the traumatic brain injury is a closed brain injury. A closed brain injury occurs when a non-penetrating injury to the brain occurs due to rapid back and forth movement and concussion of the brain inside the skull without disrupting the skull, resulting in subcutaneous bleeding and rupture of brain tissue and blood vessels. In another example, the traumatic brain injury is a penetrating brain injury. A penetrating or open head injury occurs when there is disruption of the skull, for example, when a bullet penetrates the brain. In another example, the nerve injury or disease is a spinal cord injury. Spinal cord injury refers to damage to any part of the spinal cord or nerves at the end of the spinal canal (cauda equina), which often results in permanent changes in strength, sensation, and other bodily functions below the injury site.In another example, the nerve damage or disease is multiple sclerosis (MS). In MS, the immune system attacks the protective sheath (myelin) that covers nerve fibers, which can result in permanent damage or deterioration of the nerves. In another example, the nerve damage or disease site is Alzheimer's disease, which is characterized by nerve cell damage. In another example, the nerve damage or disease is Parkinson's disease. In Parkinson's disease, nerve cells in the basal ganglia, the area of ​​the brain that controls movement, are damaged and / or die. In another example, the nerve damage or disease is Huntington's disease, which results in movement disorders, cognitive impairment, and psychiatric disorders with a wide range of signs and symptoms. In another example, the nerve damage or disease is amyotrophic lateral sclerosis (ALS). ALS affects nerve cells (motor neurons) that control voluntary muscle movement, such as walking and speaking. ALS causes motor neurons to gradually deteriorate and then die.

[0054] The terms "subject," "host," and "patient" are used interchangeably. As used herein, a subject is preferably a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans), most preferably a human.

[0055] In one example, the number of NPCs mixed with a composition disclosed herein and administered into the site of nerve injury or disease is 50,000 to 500,000 cells. In another example, the number of NPCs mixed with a composition disclosed herein and administered into the site of nerve injury or disease is 50,000 to 100,000 cells. In another example, the number of NPCs mixed with a composition disclosed herein and administered into the site of nerve injury or disease is 100,000 to 200,000 cells. In another example, the number of NPCs mixed with a composition disclosed herein and administered into the site of nerve injury or disease is 200,000 to 300,000 cells. In another example, the number of NPCs mixed with a composition disclosed herein and administered into the site of nerve injury or disease is 300,000 to 400,000 cells. In another example, the number of NPCs mixed with a composition disclosed herein and administered into the site of nerve injury or disease is 400,000 to 500,000 cells. In another example, the number of NPCs mixed with a composition disclosed herein and administered into the site of nerve injury or disease is about 50,000 cells. In another example, the number of NPCs mixed with a composition disclosed herein and administered into the site of nerve injury or disease is about 100,000 cells. In another example, the number of NPCs mixed with a composition disclosed herein and administered into the site of nerve injury or disease is about 200,000 cells. In another example, the number of NPCs mixed with a composition disclosed herein and administered into the site of nerve injury or disease is about 300,000 cells. In another example, the number of NPCs mixed with a composition disclosed herein and administered into the site of nerve injury or disease is about 400,000 cells. In another example, the number of NPCs admixed with a composition disclosed herein and administered into the site of nerve injury or disease is about 500,000 cells, hi another example, the number of NPCs admixed with a composition disclosed herein and administered into the site of nerve injury or disease is greater than 500,000 cells.

[0056] In one example, NPCs are mixed with a composition comprising a gel-forming molecule disclosed herein, a CCR5 antagonist, and thrombin, and then administered to the site of nerve injury or disease. In another example, NPCs are mixed with a composition comprising a gel-forming molecule and a CCR5 antagonist disclosed herein, and then administered to the site of nerve injury or disease, followed by administration of thrombin into the site of nerve injury or disease. Thrombin may be added separately and subsequently to the administration of the mixture of NPCs and a composition comprising a gel-forming molecule and a CCR5 antagonist disclosed herein to avoid premature gel formation prior to transplantation.

[0057] As used herein, the term "administering" and grammatical variants of this term refer to implanting a mixture of NPCs and a composition disclosed herein at the site of nerve damage or disease in a subject as disclosed herein. In one example, the mixture of NPCs and a composition disclosed herein is administered by stereotactic injection, which allows for direct injection of the NPCs at the site of nerve damage or disease as disclosed herein.

[0058] Cell therapy for neuronal injury or disease, such as stroke, traumatic brain injury, spinal cord injury, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, hypoxia-induced neuronal cell injury such as that seen in cardiac arrest or neonatal distress, neurological conditions associated with cancer, and neurodegenerative diseases, requires coordinated and dynamic regulation of transplanted NPCs, including maximal survival, adequate proliferation, neuronal-to-glial differentiation, and synaptogenesis. Advantageously, the compositions disclosed herein, when used in combination with NPCs, support the survival and differentiation of NPCs into mature neurons after transplantation of NPCs into the site of neuronal injury or disease. High levels of CCR5 have been found to be expressed on NPCs, and CCR5 antagonists promote NPC survival by blocking CCR5 signaling. Additionally, gel-forming molecules form gels to hold transplanted NPCs together and prevent them from "swimming" within the injury cyst or cavity. In the presence of fibrinogen and maraviroc, transplanted NPCs are uniformly distributed without cluster formation and differentiate into mature neurons approximately 30 days after transplantation.

[0059] In another aspect, the present disclosure refers to a kit for use in supporting the survival and differentiation of NPCs transplanted into a site of neural injury or disease, the kit comprising: (a) a composition disclosed herein; (b) artificial cerebrospinal fluid (a-CSF); (c) CaCl2; and (d) thrombin.

[0060] In one example, the kit further comprises carriers, diluents, and adjuvants for administering the mixture of NPC and a composition disclosed herein. The carriers, diluents, and adjuvants must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0061] Examples of pharmaceutically acceptable carriers or diluents are demineralized or distilled water; physiological saline solution; vegetable oils such as peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil, arachis oil or coconut oil; silicone oils including polysiloxanes, such as methylpolysiloxane, phenylpolysiloxane, and methylphenylpolysiloxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methylcellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose or are hydroxypropyl methylcellulose; lower alkanols such as ethanol or isopropanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols such as polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrrolidone; agar; gum tragacanth or gum acacia, and petrolatum. Typically, the one or more carriers form 10% to 99.9% by weight of the composition.

[0062] As used in this application, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a primer" includes a plurality of primers, including mixtures and combinations thereof.

[0063] As used herein, the term "comprising" means "including." Variations of the word "comprising," such as "comprise" and "comprises," have correspondingly different meanings. Thus, for example, a composition "comprising" X may be exclusive of X, or may include one or more additional unrecited ingredients.

[0064] As used herein, the term "about," in the context of a concentration of a substance, a size of a substance, a length of time, or other stated value, means + / - 5% of the stated value, or + / - 4% of the stated value, or + / - 3% of the stated value, or + / - 2% of the stated value, or + / - 1% of the stated value, or + / - 0.5% of the stated value.

[0065] Throughout this disclosure, certain embodiments may be disclosed 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 an inflexible limitation on the scope of the intended disclosure. Accordingly, the description of a range should be considered to have specifically disclosed not only each possible subrange, but also each individual numerical value within that range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed each individual number within that range, such as 1, 2, 3, 4, 5, and 6, as well as subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. This applies regardless of the breadth of the range.

[0066] The invention illustratively described herein may suitably be practiced in the absence of any one or more elements or limitations not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," and "containing" are to be read expansively and without limitation. Additionally, the terms and expressions used herein are used as descriptive and non-limiting terms, and the use of such terms and expressions is not intended to exclude any equivalents of the indicated and described features or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, while the invention has been disclosed in detail with preferred embodiments and optional features, it should be understood that modifications and variations of the invention disclosed herein may be employed by those skilled in the art, and that such modifications and variations are considered to be within the scope of the invention.

[0067] The invention has been described broadly and generically herein. Each of the narrower groupings of species and subgenera falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation that removes any subject matter from that genus, regardless of whether the removed material is specifically set forth herein.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0069] Other embodiments are within the scope of the following claims and non-limiting examples. [Example]

[0070] Non-limiting examples of the present disclosure will be further disclosed in more detail by reference to specific examples, which should not be construed as limiting the scope of the disclosure in any way.

[0071] Example 1 method cell culture Human embryonic stem cells (ESCs, eGFP H9 hESC line) were cultured in feeder-free mTeSR medium supplemented with 1x mTeSR supplement, 1x non-essential amino acids (NEAA), and 1x Glutamax in Matrigel-coated 6-well plates. ESCs were fed daily and passaged twice a week.

[0072] Forebrain glutamatergic NPCs were induced using the following procedure. Briefly, H9 hESCs or eGFP H9 hESCs were cultured on Matrigel / vitronectin-coated 6-well plates for 1 week. On day 0, ESC colonies were gently blown off with a 1 mL pipette to form cell aggregates. The cell aggregates were cultured in flasks for 7 days in neural induction medium (NIM) consisting of DMEM / F12, 1x N2 supplement, 1x NEAA, 2 μM SB431542, and 2 μM DMH-1. The cell aggregates were allowed to adhere to the 6-well plates for 6 hours in the presence of NIM supplemented with 5% FBS and then replaced with fresh NIM. The aggregates were fed with NIM until neural rosettes formed on day 16. The rosettes were gently blown off with a 1 mL pipette and suspended in NIM in the flasks for 7 days. From day 23, the NIM was replaced every 4 days. Neural progenitor cells (NPCs) were maintained in NIM until transplantation or immunostaining. On day 49, NPCs were disaggregated into single cells using TrypLE for 3 minutes. After an additional day of culture in NIM supplemented with 1x B27 and 100 nM Compound E, NPCs were harvested and transplanted into an ischemic stroke model. B-27 supplement is an undefined complex mixture of antioxidant enzymes, proteins, vitamins, and fatty acids, mixed in ratios optimized to support neuronal survival in culture. Compound E is a γ-secretase inhibitor. For immunostaining, NPCs were seeded onto glass coverslips and stained after 1 week of culture.

[0073] To examine the effects of CCR5 activation on NPCs and neurons, cells were seeded on Matrigel-coated coverslips or 6-well plates and treated with excess CCR5 ligands (300 ng / mL). CCL3, CCL4, CCL5, and their combination (CCL3 / 4 / 5) were added every other day in NIM. After 4 days of treatment, NPCs or neurons were harvested and stained or immunoblotted.

[0074] Generation of lentivirus Human CCR5 29-mer shRNA plasmid (pRS_hU6_CCR5shRNA_SV40_Puro) and non-effective 29-mer scrambled shRNA cassette in pRS Vector were obtained from OriGene (CAT#: TR314126, CAT#: TR30012). Lentiviral shRNA was produced in HEK 293FT cell line by transfecting the packaging and backbone plasmids. HEK 293FT cells were cultured in DMEM supplemented with 10% FBS. After 3 days of culture, the supernatant was collected. Viral particles were concentrated by ultracentrifugation at 25,000 rpm for 2.5 hours at 4°C. Viral particles were resuspended in DMEM.

[0075] shRNA transduction NPC 1×10 ^5 Cells were seeded onto glass coverslips in each well of a 24-well plate for 2 days and incubated at 37°C in a humidified 5% CO2 incubator to achieve 50% confluency at the time of transduction. NPCs were infected overnight at 37°C with lentiviral shRNA (MOI 20). The medium containing lentiviral particles was removed from the wells and replaced with 500 μL of fresh prewarmed NIM. Five days after transduction, infected NPCs were harvested for immunostaining or Western blotting.

[0076] Cocktail / composition preparation Stock solutions of 30 mg / mL fibrinogen, 50 mg / mL maraviroc, and 250 mM CaCl2 (100x) were prepared as follows: Fibrinogen (F3879, Sigma) was dissolved in a-CSF for 1 hour at room temperature. Maraviroc was dissolved in dimethyl sulfoxide (DMSO). CaCl2 (Sigma) was dissolved in deionized (DI) water. All solutions were sterile filtered and stored at -20°C until use. The cocktail / composition was generated from 2.5 mM CaCl2 with 50 mg / mL maraviroc and 30 mg / mL fibrinogen in a 1:9 volume ratio.

[0077] Release profile of maraviroc in cocktail gels By scanning over the UV range from 200 nm to 400 nm, the wavelength of maximum absorbance (λmax) of maraviroc in phosphate buffer (pH 7.4) was found to be 210 nm. A standard maraviroc drug solution was prepared by dissolving 50 mg of pure maraviroc in phosphate buffer 7.4 and transferring it into a 5 mL volumetric flask to obtain a 10 mg / mL stock solution. This solution was used as a dilution standard, from which desired solution concentrations were prepared. Final concentrations were 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0 mg / mL, and absorbances were obtained at λmax of 210 nm using appropriate blanks. Gelation of 200 μL of fibrinogen (9 mg / mL) was induced in vitro with thrombin (50 U), and the gel containing 1 mg of maraviroc was incubated in 1 mL of PBS at pH 7.4 and 37°C. For the free drug group, 5 mg of maraviroc was directly dissolved in 1 mL of PBS. 50 μL of the solution was sampled and the absorbance was examined using a Microplate Absorbance Spectrophotometer (Bio-RAD, xMark®). The final concentration of maraviroc in PBS was calculated according to a calibration curve.

[0078] Stroke model and cell transplantation All animal studies were conducted in accordance with the Institutional Animal Care and Use Committee of Duke-NUS Medical School. Ischemic stroke was induced in adult (10–12 weeks) male SCID mice by photothrombosis. Briefly, rose bengal was administered intravenously at a dose of 0.1 mg / g per mouse. The mouse skull was exposed under 2% isoflurane anesthesia. A 2.5 mm diameter cold light was applied through the intact skull to the right motor cortex (anteroposterior [AP] = +2 mm, lateral [L] = +1 mm) for 15 minutes. Animals were randomly grouped and transplanted with forebrain glutamatergic progenitor cells. Fifty thousand cells were resuspended in 1 μl of artificial cerebrospinal fluid (a-CSF) in the presence or absence of maraviroc (5 mg / mL) or fibrinogen (10 mg / mL) and injected into the injury site ([AP] = +2 mm, [L] = +1 mm, vertical [V] = -1.5 mm from the dura).

[0079] Tissue preparation and immunohistochemistry Animals were sacrificed with a lethal dose of pentobarbital (250 mg / kg) and immediately perfused with PBS followed by 4% cold paraformaldehyde (PFA). Brain samples were fixed in cold PFA for 2 hours and then immersed in 30% sucrose at 4°C for approximately 4 days until sunk. Serial coronal sections (1.54 mm to 0.22 mm from bregma) were taken on a frozen microtome at a thickness of 40 μm and stored at -20°C. For immunostaining, sections were incubated with a blocking solution containing 10% normal donkey serum and 0.2% Triton 100 for 1 hour at room temperature. Sections were then incubated with primary antibodies overnight at 4°C.

[0080] [Table 1A]

[0081] [Table 1B]

[0082] Subsequently, sections were washed and incubated with the corresponding secondary antibody for 1 hour at room temperature. Immunolabeled sections were mounted with Fluoromount-G containing Hoechst. For TUNEL staining, cells on coverslips were fixed in PBS containing 4% PFA for 30 minutes and then incubated with terminal deoxynucleotidyl transferase (TdT) equilibration buffer (Elabscience) for 30 minutes at 37°C. NPCs were incubated in labeling solution containing TdT enzyme (Elabscience) for 1 hour at 37°C.

[0083] Imaging and cell quantification To quantify the DCX, SOX2, Ki67, NF, and NeuN-positive cell populations (co-labeled with eGFP and Hoechst) relative to the total transplanted cells, one brain slice out of every six consecutive slices was selected and stereologically counted using Stereo Investigator software (MBF Bioscience) on a Zeiss M1 microscope. Briefly, immunolabeled slices were scanned on a Zeiss M1 microscope, the transplanted area was manually outlined, and the corresponding fluorescently labeled cells were then unbiasedly counted. The cleaved caspase 3-expressing cell population relative to the total transplanted cells was counted using ImageJ software. Data were collected from four to six replicates per group. The cell populations expressing Brn2, Ctip2, and Foxp2 on the coverslip were compared to the total GFP-positive population. + To quantify the cells, all coverslips were scanned and captured using a confocal microscope (Nikon) with a 20x objective, then counted using ImageJ software. Data were replicated three times. All data are presented as mean ± SEM.

[0084] Infarct area analysis and quantification The infarct area was defined by staining with GFAP, S100β, CSPG, and Iba1. The fluorescence intensity in the infarct area was measured using ImageJ software and normalized to the surrounding intact area. To measure the thickness of the infarct area, six slices were randomly selected from 35 brain slices and captured using a confocal microscope (Nikon). The perpendicular distance from the surface to the corpus callosum in the epicenter was measured using ImageJ software. All data were replicated four to six times and are presented as the mean ± SEM.

[0085] Behavioral testing Mice (n = 9–10 per group) were tested on the rotarod and grid-walk tasks. Behavior was assessed on days -14, 0, 14, and 30 after transplantation. For the rotarod test, motor function was assessed by calculating the latency to fall. For the grid-walk test, disability was calculated as the number of limb injuries (right paw) within 10 min.

[0086] Isolation of injured tissue On days 2, 14, and 44 after stroke, animals were anesthetized and perfused with cold PBS. The injured cortex or grafts (1 mm radius from the epicenter) were manually sectioned under a stereomicroscope (Zeiss) and stored at -80°C.

[0087] Western blot NPCs and neurons were washed using PBS and resuspended in RIPA buffer supplemented with protease and phosphatase inhibitors. Samples were collected in 1.5 mL tubes on ice for 15 minutes. For tissue samples, extracts were sonicated in cold RIPA buffer supplemented with protease / phosphatase inhibitors. All samples were quantified using the Quick Start® Bradford Protein Assay (Bio-RAD), and Laemmli buffer (Bio-RAD) was then added to each tube. Samples were heated at 95°C for 5 minutes and stored at -80°C. A total of 15 μg of extract was loaded per well onto a 10% Bis-Tris precast gel for electrophoresis.

[0088] After running at 120V for 40 minutes, proteins were transferred to a PVDF membrane at 400mA for 30 minutes. Subsequently, the membrane was blocked for 1 hour in 0.1% TBS-Tween (TBST) with 5% nonfat milk. The membrane was then incubated overnight at 4°C with primary antibodies: anti-CCR5 (1:1000, Abcam, ab110103), anti-CCL3 (1:1000, Abcam, ab259372), anti-CCL4 (1:1000, Abcam, EP521Y), anti-CCL5 (1:1000, Thermo Fisher Scientific, 701030), and anti-GAPDH (1:5000, Thermo Fisher Scientific, MA5-15738). The membrane was washed three times with 0.1% TBST and then incubated with the corresponding secondary antibodies at room temperature for 1 hour. Protein bands were revealed using Enhanced Chemiluminescence Substrate (Promega) and visualized using a Bio-RAD Chemidoc system. All band intensities were analyzed using ImageJ software and normalized to the corresponding GAPDH band.

[0089] Quantitative and statistical analysis Data from the stroke and transplant groups were normally distributed. Comparisons were made between the two groups using an unpaired t-test. Error bars in all figures represent the mean ± SEM. Differences at a P value of less than 0.05 were considered statistically significant. All data were analyzed using GraphPad Prism.

[0090] Example 2 result The combination of fibrinogen and a CCR5 antagonist protects transplanted NPCs from apoptosis in the ischemic core The cell therapy in this study targeted chronic stroke, as spontaneous recovery is no longer expected at this stage. During the chronic phase of stroke, the infarcted area forms a cystic or cavitary lesion surrounded by a wall of glial scar tissue and filled with inflammatory cells and secretions. This environment not only lacks physical and nutrient support but also exerts an inflammatory effect on the transplanted NPCs. To modify the hostile environment in the infarct core, we developed a cocktail / composition consisting of maraviroc (5 mg / mL) and fibrinogen (9 mg / mL). Maraviroc is an FDA-approved CCR5 inhibitor that specifically blocks the binding between CCR5 and its ligand. To slow the release of maraviroc, it was mixed with a hydrogel. Fibrinogen (9 mg / mL) was selected because it remains dissolved above 0 degrees Celsius, facilitating implantation. Upon injection, fibrinogen, mixed with endogenous thrombin, was released during surgery to form a gel. As shown by the release profile of maraviroc (Figures 1a and 6a), such injectable gels not only stabilized the transplanted cells but also slowed the dilution of maraviroc. The effects of the cocktail / composition were evaluated by directly transplanting eGFP-H9-derived cortical NPCs (differentiated from human embryonic stem cells (hESCs), eGFP-H9 hESC line, for 50 days, Figures 6c-f) into the ischemic core 2 weeks after stroke in the presence or absence of maraviroc, fibrinogen, or the cocktail / composition. The survival rate of the transplanted cells was then measured 1 week later (Figures 1b and 1c). When ischemic stroke was induced by photothrombosis in the cerebral cortex of SCID mice, without treatment, the ischemic cavity was filled with GFAP at 14 days. + and S100β + The ischemic lesions were surrounded by a glial scar (Figure 1c, Figures 6b-h), and the cortex collapsed at 30 days (Figure 6i). The ischemic lesions induced by photothrombosis exhibited a relatively uniform size in similar locations without penumbra, providing a consistent model for evaluating the efficacy of cell replacement therapy.

[0091] Transplanted cells identified by GFP were observed within the stroke cavity in the presence of fibrinogen alone or in the presence of the cocktail / composition, but not in the presence of GFP. +Few or no cells were observed in the presence of maraviroc alone or in the cell-only group (Fig. 1c and Fig. 7a). Immunostaining for cleaved caspase 3 resulted in intense fluorescence in the control, maraviroc, and fibrinogen groups, diffuse staining in the control and maraviroc groups, and individual GFP cells in the ischemic core in the fibrinogen group. + It was clear that the staining for the cells was dispersed (Fig. 1d and Fig. 1e), and the transplants in the control and maraviroc groups (GFP + ) Cells die (fragment) and individual GFP fragments form in the fibrinogen group. + In contrast, the cocktail / composition group showed no GFP positive for caspase. + There were very few cells (Fig. 1d and Fig. 1e). Thus, the transplanted NPCs survived in the presence of the cocktail / composition.

[0092] Stereological quantification of GFP (overlay against DAPI-labeled nuclei) was 1.2 × 10 in the cocktail / composition group. 5 The presence of SOX2 cells was demonstrated (Fig. 1f and 1g). Over 26% and 81% of the transplanted cells were SOX2 + and DCX + These results indicate that most NPCs begin to differentiate into neurons at an immature stage (Fig. 1f and 1g). Immunostaining for Ki67, a cell proliferation marker, revealed that Ki67 + In the cocktail / composition group, GFP + Approximately 11% of the cells were transfected with fibrinogen, whereas almost none were transfected with the other three groups (Figures 7b and 7c). Collectively, the results indicated that fibrinogen or maraviroc alone was insufficient to improve the survival of transplanted NPCs. In contrast, the combination of fibrinogen and maraviroc supported the survival of NPCs transplanted into the ischemic core.

[0093] Surviving NPCs become mature neurons The environment of the ischemic core is generally inhibitory to the differentiation of transplanted NPCs. To determine whether transplanted NPCs survive long-term and what their fate is, the transplanted brains and the number and fate of transplanted NPCs were evaluated 30 days after transplantation (Figure 2a). Macroscopically, brains from the sham (non-transplanted stroke), control, maraviroc, and fibrinogen groups showed cortical collapse and GFP expression within the ischemic region. + Although the cocktail / composition group brains had few or no cells (Fig. 8a), they displayed a smooth surface similar to that of the contralateral side (Fig. 2b and 2c). The stroke cavity is surrounded by a wall of glial scar tissue and can be tracked using GFAP and / or S100β staining. GFP-labeled transplanted cells were observed to be precisely implanted in the ischemic core and surrounded by a GFAP+ and S100β+ glial scar (Fig. 2b), suggesting that transplanted neurons do not migrate into the peri-infarct region. Remarkably, one month after transplantation, transplanted GFP+ cells filled the entire stroke cavity (Fig. 2b, 2c). Serial coronal brain sections showed that transplanted cells, confirmed by positive staining for the human-specific marker STEM121, completely filled the infarct region (Fig. 2d), and GFP + It was further revealed that less than 0.4% of the cells expressed Ki67 (Figures 8b and 8c), indicating that the majority of transplanted NPCs had stopped proliferating by 1 month without overgrowth.

[0094] Immunostaining for mature neuronal markers showed that over 89% of the transplanted cells expressed neurofilament (NF) (Fig. 2e-g). Similarly, 56% of the transplanted human (STEM121+) cells were positive for another maturation marker, NeuN (Fig. 2h, 2i). The proportion of NeuN+ / STEM121+ cells was observed to form a gradient, with NeuN+ cells more abundant in the upper layers and at the edge of the stroke cavity than in the center of the transplant (Fig. 2j-m). GFP + Approximately 9% of the cells expressed SOX9, a marker for astroglia or their precursor cells (Figs. 8d and 8e). +The majority of cells were confined to the border zone of the stroke cavity (Fig. 2e), suggesting that the transplanted neurons did not migrate into the peri-infarct region. + ) 56% of the cells were positive for NeuN, another neuronal marker (Figure 2i). The proportion of NeuN+ / STEM121+ cells was observed to form a gradient, with NeuN+ cells in the upper layers and at the edge of the stroke cavity compared to the center of the graft (Figures 2j-2m). NF is first expressed corresponding to the initiation of axonal elongation. NeuN is widely used to identify mature neurons with mature axons. Therefore, NF is expressed earlier by neurons compared to NeuN. NF and NeuN are specifically expressed by neurons, but not glial cells. Although neurons were localized to the ischemic cavity, their neurites extended into the non-injured area adjacent to the injury site, as indicated by positive staining for the human marker STEM121 and the glutamatergic neuron marker vGluT1 (Figures 9a and 9b). Furthermore, STEM121+ neurites co-expressed the presynaptic marker synapsin and the postsynaptic marker PSD95 (Figure 9c), demonstrating that the transplanted cells developed into mature neurons and formed synapses with host neurons. Collectively, these results demonstrated that human NPCs developed into mature neurons within 30 days in the presence of the cocktail / composition.

[0095] To evaluate whether cell transplantation contributes to functional improvement, behavioral tests, including the rotarod test and grid-walk test, were performed. The rotarod test showed an improvement in the fall latency of the cocktail group (Figure 10a), whereas the grid-walk test showed no significant difference between the cocktail group and the control group (Figure 10b). Therefore, partial functional recovery is achieved one month after NPC transplantation using the cocktail.

[0096] Successful transplantation is associated with attenuated glial response and restored angiogenesis In chronic stroke, glial scar tissue forms around the cavity, often causing cortical collapse. Mice that received transplants without the cocktail / composition showed cortical collapse and GFP expression 30 days after transplantation. + Few or no cells were present (Fig. 3a). There was a strong glial reaction within and around the ischemic area, as evidenced by strong staining for the microglial marker Iba1 and the astrocyte marker GFAP (Fig. 3a and 3b). Iba1+ microglia / macrophages were present in both the lesion site and penumbra, and exhibited an amoeboid morphology (Fig. 3b and 3d). GFAP + Reactive astrocytes accumulated around the infarct periphery and at the infarct interface (Figures 3b and 3e), consistent with robust deposition of chondroitin sulfate proteoglycans (CSPGs) around the ischemic area (Figures 3c and 3f). In contrast, in mice treated with the cocktail / composition, the ischemic area was markedly enriched with GFP. + The ischemic core did not collapse because it was filled with cells (Fig. 3a and 3g). More importantly, activated microglia and reactive astrocytes substantially did not accumulate within and around the ischemic area (Fig. 3d and 3e). Microglial and astrocytic processes became sparse (Fig. 3b). CSPG expression levels were significantly reduced in the border zone of the cavity (Fig. 3c and 3f), indicating a reduction in the glial scar.

[0097] In the peri-infarct region, vascular remodeling contributes to neuronal survival after ischemic stroke. Angiogenesis may also be important for transplanted cells. Immunostaining for laminin, a membrane protein that accumulates in blood vessels, revealed that blood vessels penetrated into the graft 30 days after transplantation (Fig. 3h). Compared with the other four groups, in which the vasculature was limited to the lesion edge (Fig. 8f), the vascularized area of ​​the graft showed a density comparable to that of the intact cortex (Fig. 3i), suggesting that the graft reconstituted the ischemic cavity with neovascularization. These results indicated that successful graft survival was accompanied by a reduced inflammatory response and glial scarring, as well as a restored neovascularization.

[0098] NPC transplantation with cocktail / composition down-regulates CCL and CCR5 expression Ischemic injury induces an inflammatory response, including the production of cytokines and chemokines. These receptors, including CCR5, are expressed in mature neurons within the peri-infarct region after stroke. CCR5 is one of the receptors for chemokine ligand 3 (CCL3), CCL4, and CCL5. CCL3 and CCL4 are two protein components of macrophage inflammatory protein (MIP) 1, also known as MIP1 alpha and MIP1 beta, respectively. Maraviroc, an antagonist of the chemokine receptor CCR5, has been shown to protect mature neurons separating the infarcted region but not the ischemic core. Western blot analysis showed that the expression levels of CCR5 and the three ligands were upregulated in the infarcted region after stroke (Figures 4a and 4b). The elevated levels of CCR5 and its ligand persisted 44 days after stroke, suggesting that CCR5 is persistently activated by high concentrations of ligand in the chronic infarcted area (Figs. 4b-4f).

[0099] The next question was whether maraviroc alone, fibrinogen alone, or both maraviroc and fibrinogen modulated the expression of CCR5 or CCL. Western blot analysis of transplanted cortical tissue 44 days after ischemic injury or 30 days after transplantation (Figure 4g) showed a significant decrease in CCR5 levels in the cocktail / composition group (Figures 4h and 4l). CCL3, 4, and 5 levels showed no obvious difference in the presence of maraviroc alone or fibrinogen alone, but were substantially reduced in the presence of the cocktail / composition of maraviroc and fibrinogen (Figures 4h, 4i, 4j, and 4k). The results showed that maraviroc alone or fibrinogen alone did not downregulate CCR5 or CCL3, 4, and 5 levels, whereas transplantation with the cocktail / composition significantly reduced the presence of CCL, suggesting that signaling between CCR5 in the injured cortical tissue and CCL produced by inflammatory cells was blocked.

[0100] Blockade of CCR5 activation feedback attenuates apoptosis in NPCs Studies have shown that CCR5 is upregulated in neurons within the penumbra region after stroke, and that blocking CCR5 signaling by genetic means or with maraviroc (100 mg / kg, ip daily) promotes the survival of such neurons and their synaptic connections, thereby enhancing the behavioral recovery of the animals. This raises the question of how maraviroc protected NPCs transplanted into the ischemic core. Immunostaining for neuronal differentiation using CCR5 alone revealed that CCR5 inhibits SOX2 + NPC and DCX + Although highly expressed on the membrane and cytoplasm of immature neurons (day 7), the fluorescent signal was significantly reduced in mature neurons (day 60) (Figures 5a-5c). This was confirmed by Western blot analysis, which showed a gradual decline in CCR5 expression (Figure 5d). This result suggested that NPCs and immature neurons are potentially sensitive to inflammatory cytokines present in and around the ischemic cavity.

[0101] NPCs were then incubated with three ligands (CCL3, CCL4, and CCL5, 300 ng / mL). Compared to the control group, cleaved caspase 3 was significantly increased. + The percentage of cells increased in all three groups (Fig. 11a and 11b). To mimic the environment of ischemic infarction, NPCs were incubated with a combination of three ligands (100 ng / mL of each of the three chemokines). After treatment, apoptotic neurons and detached neural NPCs were observed (Fig. 11b and 11c). Interestingly, incubation of differentiating NPCs with the three ligands from day 2 to day 4 induced upregulation of CCR5 in the cells (Fig. 5e and 5f), and TUNEL + The proportion of cells corresponding to the chemokines increased correspondingly (Fig. 5j and 5k). This result suggested that these chemokines indeed promoted CCR5 expression on NPCs and induced apoptosis of NPCs even in the absence of microglia.

[0102] The next step was to investigate whether blocking chemokine signaling could attenuate apoptosis in NPCs. We knocked down CCR5 expression using lentivirus expressing CCR5-shRNA. As shown by immunostaining and Western blot analysis, expression of CCR5-shRNA, but not control-shRNA, significantly reduced CCR5 expression in NPCs (Figures 5g-5i). Correspondingly, TUNEL + The cell population was significantly reduced in cultures treated with lentivirus carrying CCR5-shRNA (Figures 5j-5k and 12a). Similarly, blocking CCR5 with maraviroc did not reduce CCR5 expression, but rather TUNEL + The proportion of NPCs was reduced (Figures 12a and 12b). Therefore, CCR5 expression on transplanted NPCs may be amplified by the inflammatory environment of the ischemic cavity, and blockade of the CCR5 pathway, for example, by maraviroc or RNA interference (RNAi), may protect susceptible NPCs from apoptosis (Figure 5l).

[0103] Stroke-bearing mice transplanted with NPCs recovered from motor deficits Human ESC-derived neural progenitor cells were transplanted into the injury site 14 days after stroke. In rodents, this corresponds to the chronic phase in humans. This study demonstrated that the transplanted cells survived and differentiated into neurons 1 month after transplantation. The presence of human axons was observed in the brainstem and spinal cord long after transplantation. Correspondingly, stroke-affected animals recovered from motor deficits. By 12 months after transplantation, transplanted human neurons remained in the stroke area, but axons labeled with human-specific neural cell adhesion molecule (hNCAM) extended into the striatum (Figure 13D), penetrated the internal capsule (Figure 13E), and extended along the pyramidal tract (Figure 13B). Upon reaching the medulla, human axons crossed over to the contralateral side of the pyramidal tract (Figure 13C). In the spinal cord, human axons continued down the contralateral pyramidal tract to chest level. This was evident from the positive immunostaining of human neurons in the pyramidal tract and adjacent areas (T1) (Fig. 14). These results demonstrated that transplanted human cortical neurons not only survived and matured at the stroke site, but also proliferated axons along the corticospinal tract down to the spinal cord, thereby reconstructing the cortex-to-spinal tract damaged by stroke. This explains why stroke-affected animals recover from motor deficits.

[0104] Example 3 Consideration A cocktail / composition consisting primarily of maraviroc and fibrinogen was developed to support the survival of NPCs transplanted into the ischemic core. In the presence of the cocktail / composition, human NPCs transplanted into the ischemic cyst survived, then divided and matured, reconstituting the damaged cortex in a stroke model by 30 days. This was achieved by blocking signaling between inflammatory cytokines within the ischemic lesion and high levels of CCR5 on NPCs. The survival and maturation of NPCs transplanted into the ischemic core was accompanied by a significant attenuation of the glial scar and vascularization of the graft. Specifically, NPCs in the cocktail / composition group survived when quantified 7 days after transplantation. Over the next 3 weeks, surviving NPCs proliferated and filled the stroke cavity. Importantly, most of the transplanted NPCs differentiated into NeuN-positive neurons by 1 month after transplantation. In the fibrinogen-only group, there were some surviving cells that did not proliferate and became GFAP-positive astrocytes. These results demonstrated that the cocktail / composition supported the survival of transplanted NPCs in an otherwise hostile environment. Additionally, the host endothelium penetrated into the transplanted tissue, forming vasculature within the graft and supporting the long-term survival and maturation of transplanted neurons. Furthermore, glial scarring, indicated by elevated GFAP expression at the border zone of the cavity, was significantly reduced. By six months after transplantation, the transplanted neurons had projected axons not only to the brainstem and spinal cord but also to the contralateral cortex, reconnecting the ischemic cortex with the rest of the brain.

[0105] This was demonstrated to be achieved by the cocktail / composition disclosed herein, which supported NPC survival in inflammatory cavitary lesions and promoted the differentiation of surviving NPCs into neurons. Fibrinogen was able to form a gel and act as a scaffold to stabilize transplanted cells at the injury site. This also supported the proliferation of transplanted NPCs. Maraviroc, a CC chemokine receptor type 5 (CCR5) antagonist, blocked signaling between inflammatory cytokines within the ischemic lesion and high levels of CCR5 on NPCs, thereby blocking the activation of CCR5 expressed by NPCs, reducing apoptosis and promoting the maturation of transplanted NPCs. The survival and maturation of NPCs transplanted into the ischemic core was accompanied by a significant attenuation of glial scarring and vascularization of the grafts.

[0106] After ischemic stroke, inflammatory cells infiltrated the lesion and released proinflammatory cytokines, such as CCL. Reactive glial cells formed a wall around the lesion site to prevent the overflow of proinflammatory mediators. Therefore, the inflammatory environment of the ischemic cavity persisted as observed. Consequently, few NPCs transplanted into the cavity survived, and surviving NPCs tended to differentiate into astrocytes. The inflammatory pathways that induce the death of NPCs transplanted into the ischemic core are unknown. In the penumbra, CCR5 inhibits the expression of PKA and CREB on neurons, leading to increased dendritic spine loss and neuronal death. However, this does not explain the poor survival of NPCs transplanted into the ischemic core. In this study, we found that NPCs expressed high levels of CCR5, highlighting their susceptibility to the inflammatory environment. The CCL3 / 4 / 5 ligands secreted by infiltrating blood-borne cells during stroke activate microglia and astrocytes. Furthermore, reactive astrocytes and activated microglia produce CCL3 / 4 / 5, along with other cytokines, forming a cascade of inflammatory responses. Collectively, these induce apoptosis of transplanted NPCs expressing the receptor CCR5. To exacerbate this, CCLs present in the inflammatory environment further stimulate CCR5 expression. This explains why NPCs transplanted into the ischemic cavity rarely survive. In fact, no survival of NPCs transplanted into the ischemic core was observed. Therefore, existing experimental cell transplantation therapies primarily target healthy brain regions adjacent to the ischemic lesion, avoiding the toxic environment of the ischemic lesion. However, transplantation into the penumbra generates additional damage and poses substantial risks to patients. Therefore, there is a need to develop methods to protect NPCs transplanted within the inflammatory ischemic lesion. The cocktail of the present invention protects transplanted NPCs directly and indirectly by blocking the CCL-CCR5 pathway not only directly on NPCs but also on reactive glia, thereby reducing CCL production.

[0107] Protection of transplanted NPCs has primarily focused on neurotrophic support, such as increased SUMO expression in NPCs, hypoxia treatment, co-transplantation, and the use of biomaterials cross-linked with growth factors. However, these methods limit the number of viable cells and are therefore unable to replenish and reconstruct the injured brain. Because inflammation is the primary cause of cell death, blocking inflammatory signaling may be sufficient to prevent cell death. Indeed, blocking CCL-CCR5 signaling in NPCs, either by genetic means (RNAi) or by a chemical CCR5 antagonist, i.e., maraviroc, has been found to be sufficient to prevent NPC apoptosis, even in vitro. A recent study showed that administration of maraviroc (100 mg / kg, i.p., daily) rescued neurons in the peri-infarct region from death. Due to the lack of blood flow in ischemic cavities, peripheral administration of drugs is unlikely to affect NPCs transplanted into cystic or cavitary lesions. Therefore, in this study, NPCs were transplanted in the presence of maraviroc, but this was not sufficient to rescue the transplanted cells. This was probably due to the rapid dilution and / or degradation of maraviroc (half-life: 14-18 hours). Indeed, maraviroc was not administered in the presence of thrombin or Ca. ++ When mixed with fibrinogen, which forms a degradable gel in the presence of Fibrinogen and slows the release of Maraviroc, transplanted NPCs survived, although fibrinogen itself was not shown to support the survival of transplanted NPCs. Fibrinogen is neurotrophic and capable of forming a gel. The fact that fibrinogen alone does not support NPC survival suggests that simple retention of transplanted cells within cystic or cavitary lesions by neurotrophic fibrinogen may not be sufficient for cell survival. In such environments, reduction of inflammatory injury may be necessary.

[0108] Viable human NPCs were observed to proliferate and fill the entire cystic or cavitary lesion within 30 days after transplantation. Remarkably, the glial scar surrounding the ischemic cavity was significantly reduced, as evidenced by a substantial decrease in GFAP and IBA1 immunoreactivity, and the grafts were vascularized, as indicated by laminin-labeled blood vessels. These tissue modifications may be the result of complex interactions between transplanted and host cells, in addition to the effects of maraviroc. Perhaps most strikingly, the vast majority of transplanted NPCs became postmitotic neurons within 30 days, as indicated by the expression of NeuN and NF. Human cortical NPCs have a tendency to differentiate into mature neurons after prolonged proliferation, which explains the proliferation of viable NPCs and the filling of the cystic or cavitary lesion within 30 days. The rapid differentiation / maturation may be due to a lack of growth factors in this environment. This is because previous studies have shown that spinal cord NPCs transplanted into cystic or cavitary lesions of injured spinal cords can maintain a progenitor state and undergo overgrowth for several months in the presence of high concentrations (1000-fold higher than physiological concentrations) of growth factors. Therefore, the cocktail / composition disclosed herein provides a basal medium for safe cell transplantation therapy that promotes the survival and differentiation of transplanted cells, i.e., NPCs. This medium may be supplemented with growth factors to promote proliferation, such as FGF2, or neurotrophic factors for further survival and maturation, such as BNDF and GDNF, or Notch inhibitors, such as Compound E, that promote cell cycle exit, depending on the number and developmental stage of donor cells and the size of the lesion to be repaired. While the basic cocktail / composition can be modified to meet the needs of the disease and the characteristics of the NPCs, the cocktail / composition disclosed herein opens the possibility of repairing gap-forming lesions, such as those caused by stroke and other inflammatory neurological conditions, through cell transplantation therapy. [Industrial Applicability]

[0109] It is apparent that various other modifications and adaptations of the present invention will become apparent to those skilled in the art after reading and understanding the foregoing disclosure without departing from the spirit and scope of the present invention, and all such modifications and adaptations are intended to be within the scope of the appended claims.

Claims

1. 1. A composition for supporting the survival and differentiation of neural progenitor cells (NPCs) transplanted into a site of nerve injury or disease, comprising: (a) a gel-forming molecule, and (b) Chemokine receptor type 5 (CCR5) antagonist A composition comprising:

2. 10. The composition of claim 1, wherein the gel-forming molecules are selected from the group consisting of fibrinogen, agarose, collagen, gelatin, chitosan, alginate, fibrin, hyaluronic acid, laminin, and degradable polymers selected from the group consisting of poly(glycolic acid) (PGA), poly(caprolactone) (PCL), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), and poly(ethylene glycol) (PEG).

3. The composition of claim 2 , wherein the gel-forming molecule is fibrinogen.

4. 4. The composition of any one of claims 1 to 3, wherein the CCR5 antagonist is a small molecule selected from the group consisting of maraviroc, fusucine, TAK-220, nifeviroc, DAPTA, aplaviroc, aplaviroc hydrochloride, ophiobolin C, AZD-5672, and maraviroc-d6, or a nucleic acid selected from the group consisting of small interfering RNA (siRNA), small hairpin RNA (shRNA), and microRNA (miRNA).

5. The composition of claim 4, wherein the CCR5 antagonist is maraviroc.

6. 6. The composition of any one of claims 1 to 5, wherein the NPCs are selected from the group consisting of NPCs derived from human embryonic stem cells (ESCs), NPCs derived from induced pluripotent stem cells (iPSCs), embryonic NPCs, and adult NPCs.

7. 7. The composition of claim 1, wherein the site of nerve damage or disease is characterized by inflammation and is a site resulting from nerve damage or disease selected from the group consisting of stroke, traumatic brain injury, spinal cord injury, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS).

8. CaCl 2 8. The composition of claim 1, further comprising:

9. 9. The composition of claim 1, further comprising thrombin.

10. 10. The composition of claim 9, wherein the thrombin is at a concentration of 10 U / mL to 500 U / mL.

11. 4. The composition of claim 3, wherein the fibrinogen is at a concentration of 5 mg / mL to 30 mg / mL.

12. 6. The composition of claim 5, wherein the maraviroc is at a concentration of 3 mg / mL to 50 mg / mL.

13. (a) mixing NPC with a composition according to any one of claims 1 to 12; (b) administering a mixture of the NPCs and the composition into the subject at the site of neural injury or disease, thereby supporting the survival and differentiation of the NPCs.

10. A method of treating a neurological injury or disease in a subject, comprising:

14. 14. The method of claim 13, wherein the NPC is selected from the group consisting of NPC derived from human embryonic stem cells (ESCs), NPC derived from induced pluripotent stem cells (iPSCs), embryonic NPCs, and adult NPCs.

15. 15. The method of claim 13 or 14, wherein the neurological injury or disease is characterized by inflammation and is selected from the group consisting of stroke, traumatic brain injury, spinal cord injury, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS).

16. The method of any one of claims 13 to 15, wherein the number of NPCs mixed with the composition is between 50,000 and 500,000 cells.

17. The method of claim 16, wherein the number of NPCs mixed with the composition is 50,000 cells.

18. 18. The method of any one of claims 13 to 17, wherein thrombin is administered to the site of nerve injury or disease after administration of a mixture of NPC and a composition of any one of claims 1 to 12.

19. 13. Use of a mixture of NPCs and a composition described in any one of claims 1 to 12 in the manufacture of a medicament for treating neural damage or disease in a subject, wherein the mixture is administered into the site of neural damage or disease in the subject, thereby supporting the survival and differentiation of the NPCs.

20. 20. The use of claim 19, wherein the NPC is selected from the group consisting of NPC derived from human embryonic stem cells (ESCs), NPC derived from induced pluripotent stem cells (iPSCs), embryonic NPCs, and adult NPCs.

21. 21. The use of claim 19 or 20, wherein the neurological injury or disease is characterized by inflammation and is selected from the group consisting of stroke, traumatic brain injury, spinal cord injury, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS).

22. The use according to any one of claims 19 to 21, wherein the number of NPCs mixed with the composition is between 50,000 and 500,000 cells.

23. 23. The use of claim 22, wherein the number of NPCs mixed with the composition is 50,000 cells.

24. 24. The use according to any one of claims 19 to 23, wherein thrombin is administered to the site of nerve injury or disease after administration of a mixture of NPC and a composition according to any one of claims 1 to 12.

25. 1. A kit for use in supporting the survival and differentiation of NPCs transplanted into a site of neural injury or disease, comprising: (a) a composition according to any one of claims 1 to 12; (b) artificial cerebrospinal fluid (a-CSF); (c) CaCl 2 , and (d) thrombin Includes a kit.

Images