Compositions for cell transplantation therapy and uses thereof

Abstract

Disclosed is a composition for supporting the survival and differentiation of neural precursor cells (NPCs) transplanted to 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 are methods of treating a nerve injury or disease in a subject comprising (a) mixing NPC with the composition disclosed herein; and (b) administering a mixture of NPC and a composition to the subject's nerve injury or disease site, thereby supporting the survival and differentiation of NPC. Also disclosed is the use of a mixture of NPC and the compositions disclosed herein in the manufacture of a medicament for the treatment of a nerve injury or disease in a subject wherein the mixture is administered to a site of the nerve injury or disease in a subject, thereby supporting the survival and differentiation of NPC. Further disclosed is a kit for supporting NPC survival and differentiation transplanted to a site of nerve injury or disease, the kit comprising: (a) a composition disclosed herein; (b) artificial cerebrospinal fluid (a-CSF); (c) CaCl2 (CaCl2); and (d) thrombin.

Description

Technical Field

[0001] The present invention generally relates to a composition for supporting cell survival and differentiation in cell transplantation therapy and uses thereof. In particular, the present invention relates to a composition for supporting the survival and differentiation of neural progenitor cells (NPCs) in NPC transplantation therapy for nerve injury or disease and uses thereof. Background Art

[0002] Nerve injury or disease, including stroke, traumatic brain injury, and spinal cord injury, is a major cause of disability for which there is no effective treatment. For example, ischemic stroke is caused by an acute reduction in cerebral blood flow and afflicts approximately 25% of people during their lifetime, accounting for almost 5% of all disability-adjusted life-years and 10% of all deaths worldwide. Conventional treatment is reperfusion in the acute phase of the ischemic event. However, there is no effective treatment beyond the acute phase. Therefore, the need to develop a treatment for nerve injury or disease, such as ischemic stroke, remains unmet.

[0003] Stem cell-based methods are promising because cell therapy can protect damaged neurons from further injury and / or replace lost neurons. Transplantation of non-neural cells, such as mesenchymal stem cells (MSCs), has been initiated in clinical trials. It has been shown that MSCs protect neurons in the penumbra region, which is the brain region at the boundary between the ischemic site and the healthy brain, by modulating inflammation or angiogenesis. However, MSC transplantation does not result in neuron replacement. Since the regenerative capacity of the human brain is very limited and MSCs generally do not produce neurons, it is desirable to use neural cells as a source of cell therapy.

[0004] In fact, 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 they can mature into functional neurons and have the potential to integrate into the host brain circuitry. In animals with nerve injury or disease such as ischemic stroke, a lesion cyst or cavity forms at the site of the nerve injury or disease (such as the ischemic / infarct site), which is isolated by a glial scar and filled with inflammatory cells and secretions. This environment is not conducive to transplanted NPCs, resulting in low survival rates of NPCs transplanted into the lesion cyst or cavity and nerves unable to grow through the glial scar. The inhibitory environment also promotes the differentiation of transplanted NPCs into glial cells rather than neurons. Efforts are being made to improve the survival rate of NPCs transplanted into the ischemic core, including overexpression of small ubiquitin-like modifier (SUMO), hypoxic treatment, co-transplantation with non-neuronal cells, and hydrogels crosslinked with growth factors (such as bone morphogenetic protein (BMP4), brain-derived neurotrophic factor (BDNF), and laminin-derived motif (IKVAV))-based biomaterials. In particular, hydrogels have an anti-inflammatory effect and can be absorbed by tissues. Studies have shown that hyaluronic acid-methylcellulose exhibits anti-inflammatory properties by reducing the level of IL-1α in the central nervous system after stroke and spinal cord injury. Hydrogels can also be modified to regulate the immune response and promote angiogenesis, potentially promoting the survival and differentiation of transplanted NPCs. When encapsulated in a hydrogel, NPCs transplanted into the stroke cavity can survive for 2 weeks, but the proliferation is limited. The anti-inflammatory polarization effect of hydrogels on infiltrating microglia may indicate the potential for inflammatory reprogramming of stroke lesions, which may contribute to nerve regeneration after NPC transplantation. However, due to the limited number of surviving cells, these methods are unable to fill and reconstruct the damaged brain. Relative to other neurological models such as the 6-OHDA-induced Parkinson model, the cell survival rate is very low even in the presence of neurotrophic support, which led to the hypothesis underlying the present invention that altering the adverse ischemic environment, especially the inflammatory environment, is crucial for promoting NPC survival.

[0005] Currently, most studies transplant NPCs into the penumbra to avoid the adverse environment in the lesion cyst or cavity. However, it causes additional damage to healthy brain regions. It also leaves the lesion cyst or cavity unfilled, especially when the ischemic cyst is relatively large, leaving the isolated brain regions unconnected. Another conventional method is to use a mixture of growth factors that support the survival of NPCs transplanted into damaged spinal cysts. However, growth factors at a thousand-fold physiological concentration are required. This does result in a large graft that fills the lesion cyst or cavity. However, it often creates tissue occupancy in the spinal cord. In addition, it prevents neural progenitor cells from differentiating into mature neurons and glia, which is necessary for the transplanted cells to integrate into the host tissue to achieve the therapeutic goal.

[0006] Transplanting NPCs into a diseased cyst or cavity may fill the gap, replace lost nerve cells, and reconnect disrupted circuits. Effective differentiation or maturation of transplanted NPCs is also crucial for cell transplantation therapy to work. Accordingly, a composition and method are needed to achieve and improve the survival of transplanted NPCs and to promote the differentiation of transplanted NPCs into mature neurons for the reconstitution of nerve injury or disease sites. SUMMARY OF THE INVENTION

[0007] The present disclosure describes a composition comprising two components, a gel-forming molecule and a C-C chemokine receptor type 5 (CCR5) antagonist. In one example, the gel-forming molecule and the CCR5 antagonist are FDA-approved drugs, fibrinogen and Maraviroc, respectively. In the presence of the composition, NPCs transplanted into a nerve injury or disease site such as an ischemic core survive and subsequently differentiate into neurons, which reconstitutes the collapsed cortex.

[0008] In one aspect, the invention relates to a composition for supporting the survival and differentiation of neural progenitor cells (NPCs) transplanted into a nerve injury or disease site, the composition comprising:

[0009] (a) a gel-forming molecule; and

[0010] (b) a C-C chemokine receptor type 5 (CCR5) antagonist.

[0011] In another aspect, the present disclosure relates to a method of treating a nerve injury or disease in a subject, the method comprising

[0012] (a) mixing NPCs with the composition disclosed herein; and

[0013] (b) administering the mixture of NPCs and the composition to the nerve injury or disease site of the subject, thereby supporting the survival and differentiation of the NPCs.

[0014] In another aspect, the present disclosure relates to the use of a mixture of NPCs and the composition disclosed herein in the preparation of a medicament for treating a nerve injury or disease in a subject, wherein the mixture is administered to the nerve injury or disease site of the subject, thereby supporting the survival and differentiation of the NPCs.

[0015] In another aspect, the invention relates to a kit for supporting the survival and differentiation of NPCs transplanted into a nerve injury or disease site, the kit comprising:

[0016] (a) the composition disclosed herein;

[0017] (b) artificial cerebrospinal fluid (a-CSF);

[0018] (c) CaCl2; and

[0019] (d) Thrombin.

[0020] Advantageously, after NPC transplantation, the gel-forming molecules in the composition form a gel at 37°C and thus serve as a scaffold to stabilize the NPCs transplanted into the diseased cyst or cavity. The gel also prevents the rapid dilution of the CCR5 antagonist. The CCR5 antagonist blocks the signal transduction from inflammatory cytokines in the diseased cyst or cavity to CCR5 expressed on NPCs, reduces apoptosis of transplanted NPCs in the inflamed diseased cyst or cavity, and promotes the differentiation of surviving NPCs. In addition, the composition contains physiologically-concentrated FDA-approved drugs, making it suitable for the clinical setting. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the drawings, in which:

[0022] Figure 1 Illustrates the survival of NPCs transplanted using the compositions disclosed herein in the ischemic core. Figure 1 a is a schematic diagram showing the process of transplanting NPCs with fibrinogen and maraviroc.

[0023] Figure 1 b is a schematic diagram showing the experimental procedures and timeline for inducing ischemic stroke, cell transplantation, and tissue harvest for analysis.

[0024] Figure 1 c is a series of fluorescence microscopy images taken using a Nikon Ti2 confocal microscope, showing the immunostaining of glial fibrillary acidic protein (GFAP) in stroke mice transplanted with NPCs in the presence of either a-CSF alone, maraviroc alone, fibrinogen alone, or the composition, on day 7 post-transplantation, showing GFP in the diseased cavity (marked with a dashed outline), + transplanted cells (green) and GFAP around the diseased cavity + glial scar. The dashed line outlines the ischemic core. Scale bar, 200 μm.

[0025] Figure 1 d is a series of fluorescence microscopy images of cortical sections taken using a Nikon Ti2 confocal microscope, showing the immunoreactivity of cleaved-caspase3 (red) in transplanted cells at 7 days post-transplantation. The individual fluorescence channels are shown below the microscopy images. Scale bar, 100 μm.

[0026] Figure 1 e shows GFP + cells with cleaved-caspase3 +Bar graph of the quantification of cell proportions. n = 4 mice per group. Data are mean ± SEM.

[0027] Figure 1 f is a series of fluorescence microscopy images taken using a Nikon Ti2 confocal microscope, showing the immunostaining of DCX and SOX2 in stroke mice transplanted with NPCs at 7 days post-transplantation. Yellow arrows indicate surviving grafts. The dashed line outlines the ischemic core. Scale bar, 200 μm.

[0028] Figure 1 g illustrates GFP + cells in DCX + or SOX2 + Dot plot of the number of cells. n = 5 mice in the mixture / composition group and n = 4 mice in all other three groups. Data are mean ± SEM.

[0029] Figure 2 Illustrates the maturation of NPCs transplanted in the ischemic core using the compositions disclosed herein. Figure 2 a is a schematic diagram showing the procedures and timeline for inducing ischemic stroke, cell transplantation, and tissue harvest for analysis.

[0030] Figure 2 b is a fluorescence microscopy image taken using a Nikon Ti2 confocal microscope, providing an overview of transplanted human cells (labeled by STEM121) in the cortical ischemic core (surrounded by GFAP + glial scar) of stroke mice transplanted with the mixture / composition at 30 days post-transplantation (30-dpt). Separate channels are shown on the right. Scale bar, 1 mm. LV, lateral ventricle. cc, corpus callosum.

[0031] Figure 2 c is a whole-brain field-of-view section of mice with (bottom panel) or without (top panel) NPC transplantation. Dashed lines and black arrows indicate the damaged or transplanted sites. Scale bar, 2 mm.

[0032] Figure 2 d shows serial coronal sections, which demonstrate that STEM121 + cells filled the stroke cavity at 30 days post-transplantation (30-dpt). Scale bar, 1 mm.

[0033] Figure 2 e and 2f are fluorescence microscopy images taken using a Nikon Ti2 confocal microscope, showing the immunostaining of neurofilament (NF), which shows the expression of NF in transplanted cells (GFP + cells) at 30-dpt. Figure 2The magnified image in f shows that GFP cells are NF positive. Scale bar, Figure 2 200 μm in e, Figure 2 100 μm in f.

[0034] Figure 2 g is a pie chart showing the quantification of the percentage of NF + cells in GFP + cells. n = 4 mice. Data are mean ± SEM.

[0035] Figure 2 h is a fluorescence microscopic image taken using a Nikon Ti2 confocal microscope, showing the immunostaining of STEM121 and NeuN, indicating that the transplanted cells differentiated into mature neurons 30 days post-transplantation (30-dpt). Scale bar, 1 mm. LV, lateral ventricle. cc, corpus callosum.

[0036] Figure 2 i is a bar chart showing the number of GFP + NeuN + or GFP + NeuN - cells. n = 5 mice. Data are mean ± SEM.

[0037] Figure 2 j - 2l are Figure 2 images of the area shown in h, showing the transplanted cells at the border ( Figure 2 j), upper layer ( Figure 2 k), and deep layer ( Figure 2 l). Scale bar, 100 μm.

[0038] Figure 2 m is a bar chart showing the quantification of the percentage of NeuN + cells in the transplanted cells in the upper and deep layers. n = 4 mice. Data are mean ± SEM. p = 0.0013. **p < 0.01.

[0039] Figure 3 The glial reaction and angiogenesis in the transplanted brain are shown.

[0040] Figure 3 a is a series of fluorescence microscopic images taken using a Nikon Ti2 confocal microscope, showing the immunostaining of Iba1 and GFAP in the brains of mice transplanted with sham-operated NPCs, a-CSF alone, maraviroc alone, fibrinogen alone, or the composition at 30 days post-transplantation. Scale bar, 200 μm. cc, corpus callosum.

[0041] Figure 3b is an enlarged view showing the different immunoreactivities of Iba1 and GFAP in mice treated with different treatments. The asterisk indicates the ischemic core. Scale bar, 200 μm.

[0042] Figure 3 c is a series of fluorescence microscopic images taken using a Nikon Ti2 confocal microscope, showing the immunostaining of CSPG and S100β, which shows the glial response in mice treated with the composition compared to other groups. The asterisk indicates the ischemic core. Scale bar, 200 μm.

[0043] Figure 3 d - 3f are bar graphs showing the quantification of the indicated fluorescence intensity (normalized to the intact region) at the lesion site 30 days after transplantation. n = 5 mice per group. Data are mean ± SEM. Figure 3 **p = 0.0017 in d, Figure 3 *p = 0.0372 in e, Figure 3 ***p = 0.0004 in f.

[0044] Figure 3 g is a bar graph showing the quantification of the parenchyma volume in the damaged area surrounded by GFAP + cells. n = 5 mice per group. Data are mean ± SEM. ***p = 0.0009.

[0045] Figure 3 h is a series of fluorescence microscopic images taken using a Nikon Ti2 confocal microscope, showing the immunostaining of STEM121 and laminin, showing angiogenesis in the graft 30 days after transplantation. Scale bar, 200 μm.

[0046] Figure 3 i is a bar graph showing the quantification of the vascular density in the graft and the intact region. n = 5 mice. Data are mean ± SEM. ns p = 0.1146.

[0047] Figure 4 Shows the changes in the expression of chemokine ligand (CCL) and chemokine receptor type 5 (CCR5) in the transplanted brain.

[0048] Figure 4 a shows Figure 4 The experimental strategy of b - 4f.

[0049] Figure 4 b is a western blot image showing the protein expression of CCR5, CCL3, CCL4, and CCL5 in the peri - infarct and infarcted cortex at 2, 14, and 44 days post - stroke (dps).

[0050] Figure 4c-4f shows the protein expression quantification bar graphs of CCL3 ( Figure 4 c), CCL4 ( Figure 4 d), CCL5 ( Figure 4 e), and CCR5 ( Figure 4 f), and these protein expressions are normalized relative to GAPDH. n = 3 mice per group. Data are mean ± SEM. Figure 4 In c, *p = 0.0136, Figure 4 in d, *p = 0.0241, Figure 4 in e, *p = 0.0499, Figure 4 in f, *p = 0.0244.

[0051] Figure 4 g shows Figure 4 the experimental strategy of h-4l.

[0052] Figure 4 h shows the Western blot images of CCR5, CCL3, CCL4, and CCL5 protein expressions in the graft and infarct regions at 44 days after stroke.

[0053] Figure 4 i-4l shows the protein expression quantification bar graphs of CCL3 ( Figure 4 i), CCL4 ( Figure 4 j), CCL5 ( Figure 4 k), and CCR5 ( Figure 4 l), and these protein expressions are normalized relative to GAPDH. In Figure 4 i-4k, n = 3 mice per group, and in Figure 4 l, n = 4 mice per group. Data are mean ± SEM. Figure 4 In i, *p = 0.0174, Figure 4 in j, **p = 0.0046, Figure 4 in k, **p = 0.0079, Figure 4 in l, **p = 0.0066.

[0054] Figure 5 The expression and regulation of CCR5 in NPCs are shown.

[0055] Figure 5 a-5c are fluorescence microscopic images taken using a Nikon Ti2 confocal microscope, showing the immunostaining of CCR5 with SOX2 ( Figure 5 a), DCX ( Figure 5 b), and NeuN ( Figure 5 c), showing the expression of CCR5 in NPCs, immature, and mature neurons. Scale bar, 200 μm.

[0056] Figure 5 d is 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 mean ± SEM relative to GAPDH. **p = 0.0094.

[0057] Figure 5 e shows Figure 5 Schematic diagrams of the experimental strategies for f - k.

[0058] Figure 5 f is a Western blot image showing the expression levels of CCR5 on NPCs with or without CCR5 - shRNA in the presence or absence of CCL.

[0059] Figure 5 g - 5h are fluorescence microscopic images taken using a Nikon Ti2 confocal microscope. They are immunostainings of SOX2 and CCR5, showing the expression of CCR5 on NPCs with vehicle ( Figure 5 g) or CCR5 - shRNA ( Figure 5 h). Scale bar, 200 μm.

[0060] Figure 5 i is a Western blot image showing the expression levels of CCR5 on the indicated cells.

[0061] Figure 5 j is a fluorescence microscopic image of NPCs taken using a Nikon Ti2 confocal microscope, showing the number of apoptotic NPCs (TUNEL + ) induced by CCL in the absence or presence of CCR5 - shRNA. Scale bar, 200 μm.

[0062] Figure 5 k is a dot plot showing the quantification of the percentage of TUNEL + cells in NPCs. n = 5 samples per group. Data are mean ± SEM. ****p < 0.0001.

[0063] Figure 5 l is a schematic diagram showing that blocking CCR5 activation feedback reduces NPC apoptosis.

[0064] Figure 6 Shows the establishment of ischemic stroke by photothrombosis and the preparation of NPCs for transplantation.

[0065] Figure 6 a is a line graph showing the in vitro maraviroc release curves of the mixture gel and free drug.

[0066] Figure 6 b is an image showing 2,3,5-triphenyltetrazolium chloride (TTC) staining on a brain slice, showing the infarct area at 3 days post-stroke (dps). Black arrows indicate the infarct area. Scale bar, 1 mm.

[0067] Figure 6 c - 6e are fluorescence microscopy images of immunostaining of cortical markers Brn2 (upper layer, c), Ctip2 (deep layer, d), and Foxp2 (deep layer, e) taken using a Nikon Ti2 confocal microscope, showing different subtypes of cortical progenitor cells for transplantation. Scale bar, 200 μm.

[0068] Figure 6 f shows GFP + Bar graph quantifying the percentage of NPCs indicated in GFP cells. n = 3 samples. Data are mean ± SEM.

[0069] Figure 6 g is an image of dissociated GFP + NPCs before transplantation. Scale bar, 200 μm.

[0070] Figure 6 h is a fluorescence microscopy image of immunostaining of astrocyte markers GFAP and S100β taken using a Nikon Ti2 confocal microscope, showing the ischemic core surrounded by reactive astrocytes at 14 - dps. Dashed line indicates the ischemic core and corpus callosum (cc). Scale bar, 200 μm.

[0071] Figure 6 i is a fluorescence microscopy image of immunostaining of neurite markers NF and microtubule-associated protein 2 (MAP2) on a cortical slice taken using a Nikon Ti2 confocal microscope, showing that the damaged cortex has collapsed at 30 - dps and has no neurites. Scale bar, 200 μm.

[0072] Figure 7 Shows the survival and proliferation of transplanted NPCs.

[0073] Figure 7 a is a fluorescence microscopy image of NeuN immunostaining taken using a Nikon Ti2 confocal microscope, showing that at 7 days after transplantation, NPCs (GFP + ) were transplanted into the ischemic core (NeuN - ). Scale bar, 200 μm. PI, perilesional. cc, corpus callosum.

[0074] Figure 7b is a fluorescence microscopic image of Ki67 immunostaining taken using a Nikon Ti2 confocal microscope, showing the expression of Ki67 in transplanted cells 7 days after transplantation. The boxed area is magnified in the figure below. Yellow arrows indicate cells co-labeled with Ki67 and GFP. Scale bar, 200 μm.

[0075] Figure 7 c is a bar graph showing the quantification of the percentage of Ki67 + cells in transplanted cells (GFP + ). n = 4 mice per group. Data are mean ± SEM.

[0076] Figure 8 The survival of transplanted cells in the ischemic core at 30 dpt is shown.

[0077] Figure 8 a is a fluorescence microscopic image taken using a Nikon Ti2 confocal microscope, showing the immunostaining of NeuN and DCX in mice transplanted with NPCs (GFP + ) in the specified medium, showing no GFP + cell survival. Scale bar, 200 μm.

[0078] Figure 8 b is an image of the graft in the mixture / composition group, showing the expression of Ki67 in transplanted cells (GFP + ) 30 days after transplantation. Scale bar, 1 mm.

[0079] Figure 8 c is a pie chart showing the quantification of the percentage of Ki67 + cells in transplanted cells. n = 4 mice.

[0080] Figure 8 d is a fluorescence microscopic image taken using a Nikon Ti2 confocal microscope, showing the immunostaining of SOX9 in mice transplanted with NPCs in the mixture / composition group 30 days after transplantation. Scale bar, 1 mm.

[0081] Figure 8 e is a pie chart showing the quantification of the percentage of SOX9 + cells in transplanted cells. n = 4 mice. cc, corpus callosum.

[0082] Figure 8 f shows the immunostaining of GFP and laminin, showing the four groups of collapsed cortices one month after transplantation. Scale bar, 500 μm. cc, corpus callosum.

[0083] Figure 9 It is illustrated that transplanted cells treated with the mixture project axons out of the ischemic core 30 days after transplantation.

[0084] Figure 9 a shows the immunostaining of STEM121 and GFAP in mice transplanted with the mixture and NPCs at 30 days post-transplantation, showing axons growing through the glial scar. Scale bar, 200 μm.

[0085] Figure 9 b shows the immunostaining of STEM121 and the glutamatergic marker VGluT1, showing the differentiation of transplanted cells into glutamatergic neurons. Scale bar, 10 μm.

[0086] Figure 9 c shows the immunostaining of STEM121, synaptophysin (presynaptic marker), and psd95 (postsynaptic marker) in the uninjured area adjacent to the injury site, showing the formation of synapses (white arrows) between neurites of transplanted neurons and host neurons. Scale bar, 20 μm. cc, corpus callosum.

[0087] Figure 10 The results of behavioral tests performed at -14 days (before stroke), 0 days, 14 days, and 30 days post-transplantation are shown.

[0088] Figure 10 a shows the quantification of the fall latency in the rotarod test, showing motor recovery at 30 days post-transplantation. n = 9 mice in the maraviroc and fibrinogen groups, and n = 10 mice in all other groups. Data are mean ± SEM. *p = 0.034 (mixture vs control).

[0089] Figure 10 b shows the motor ability data evaluated by the grid walk test. n = 9 mice in all groups in b. Data are mean ± SEM. n.s. p = 0.053 (mixture vs control).

[0090] Figure 11 CCL-induced apoptosis of NPCs is shown.

[0091] Figure 11 a is a fluorescence micrograph of the immunostaining of SOX2, STEM121, and cleaved-caspase3 taken using a Nikon Ti2 confocal microscope, showing the differential expression of cleaved-caspase3 in NPCs treated as indicated. Scale bar, 200 μm.

[0092] Figure 11 b is a bar graph that shows cleaved-caspase3 indicating apoptosis in the presence of high (300 ng / mL) and low (10 ng / mL) concentrations of CCL +Quantification of the percentage of cells. n = 5 samples for the low CCL group and n = 4 samples for the other four groups. Data are mean ± SEM. **p = 0.0058.

[0093] Figure 11 c is the differential image of live NPCs and immature neurons under the specified treatment. Scale bar, 200 μm.

[0094] Figure 12 The effect of CCR5 inhibition on NPC survival is shown.

[0095] Figure 12 a is a fluorescence microscopic image taken using a Nikon Ti2 confocal microscope, showing the fluorescence staining of CCR5, STEM121, and TUNEL in NPCs under the specified treatment. Scale bar, 200 μm.

[0096] Figure 12 b is a bar graph showing the quantification of the proportion of apoptotic cells (TUNEL + ) in NPCs in the presence of shRNA or maraviroc. n = 3 samples per group. Data are mean ± SEM. **p = 0.0064, ****p < 0.0001.

[0097] Figure 13 Immunostaining of hNCAM (human neuronal marker) in the brains of ischemic mice is shown. This indicates the projection of transplanted human neurons from the cortex to the brainstem. The corresponding magnification is shown in the right panel. Scale bar: 1 mm.

[0098] Figure 14 Immunostaining of hNCAM in the spinal cords of ischemic mice transplanted with human neurons is shown. This indicates that the axons of transplanted neurons project into the spinal cord. The corresponding magnifications are shown in Figures A and B. Scale bar: 200 μm. Detailed implementation

[0099] The present disclosure describes a composition comprising FDA-approved drugs (such as fibrinogen and the CCR5 inhibitor maraviroc), which supports the survival and differentiation of NPCs transplanted into the site of nerve injury or disease in the brain, resulting in filling the gap in the brain lesion, replacing lost cells, reconnecting damaged neural circuits, and thus achieving behavioral recovery in a subject suffering from nerve injury or disease. The composition can be used in cell transplantation therapies for nerve 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 neuropathological conditions characterized by inflammation).

[0100] In one aspect, the present invention relates 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 C-C chemokine receptor type 5 (CCR5) antagonist.

[0101] As used herein, the term "support" means to maintain, promote, increase, or enhance the proportion of NPCs that survive and differentiate into functional neurons and glial cells after transplantation into a site of nerve injury or disease.

[0102] As used herein, the term "survival" refers to the viability of a cell (in this case, an NPC), characterized by the ability to perform certain functions such as metabolism, growth, reproduction, some form of response, and adaptation. In one example, if an NPC transplanted into a site of nerve injury or disease does not express cleaved-caspase3, the NPC is viable and alive. 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 is viable and alive. In one example, in the presence of the composition disclosed herein, at least 80% of the NPCs are viable after transplantation into a site of nerve injury or disease. In another example, in the presence of the composition disclosed herein, at least 85% of the NPCs are viable after transplantation into a site of nerve injury or disease. In another example, in the presence of the composition disclosed herein, at least 90% of the NPCs are viable after transplantation into a site of nerve injury or disease. In another example, in the presence of the composition disclosed herein, at least 95% of the NPCs are viable after transplantation into a site of nerve injury or disease. In another example, in the presence of the composition disclosed herein, at least 99% of the NPCs are viable after transplantation into a site of nerve injury or disease. In another example, in the presence of the composition disclosed herein, approximately 100% of the NPCs are viable after transplantation into a site of nerve injury or disease.

[0103] As used herein, the term "differentiation" refers to changing NPCs into more specialized cell types, such as neurons and / or glial cells. In one instance, in the presence of the compositions disclosed herein, NPCs transplanted to a site of nerve injury or disease differentiate into neurons. In another instance, the differentiated neurons are selected from the group consisting of bipolar, multipolar, and pseudounipolar neurons. In another instance, the differentiated neurons are bipolar neurons that have one axon and one dendrite extending from the cell body. In another instance, the differentiated neurons are multipolar neurons that contain one axon and multiple dendrites. Multipolar neurons can be found in the central nervous system (CNS, which consists of the brain and spinal cord). In another instance, the multipolar neurons are Purkinje cells in the cerebellum, which have many branched dendrites but only one axon. In another instance, the differentiated neurons are pseudounipolar neurons that have a single process extending from the cell body, but the process later branches into two different structures, such as a bipolar cell. In one instance, the pseudounipolar neurons are sensory neurons that have an axon that branches into two extensions: one that connects to a dendrite that receives sensory information, and another that transmits that information to the spinal cord. In another instance, in the presence of the compositions disclosed herein, NPCs transplanted to a site of nerve injury or disease differentiate into glial cells. In another instance, the differentiated glial cells are selected from the group consisting of astrocytes, microglia, oligodendrocytes, radial glia, and ependymal cells. In another instance, the differentiated glial cells are astrocytes that contact capillaries and neurons in the CNS to provide nutrients and other substances to the neurons, regulate the concentration of ions and chemicals in the extracellular fluid, and provide structural support for synapses. In another instance, the differentiated glial cells are microglia that clear and degrade dead cells and protect the brain from microbial invasion. In another instance, the differentiated glial cells are oligodendrocytes that form myelin sheaths around axons in the CNS. In another instance, the differentiated glial cells are radial glia that act as scaffolds for developing neurons as they migrate to their final destinations. In another instance, the differentiated glial cells are ependymal cells that line the fluid-filled ventricles and the central canal of the spinal cord.

[0104] In one instance, in the presence of the compositions disclosed herein, NPCs differentiate into neurons after transplantation to a site of nerve injury or disease. In one instance, in the presence of the compositions disclosed herein, 85%-95% of the NPCs differentiate into neurons after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, 85%-90% of the NPCs differentiate into neurons after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, 90%-95% of the NPCs differentiate into neurons after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, approximately 85% of the NPCs differentiate into neurons after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, approximately 86% of the NPCs differentiate into neurons after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, approximately 87% of the NPCs differentiate into neurons after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, approximately 88% of the NPCs differentiate into neurons after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, approximately 89% of the NPCs differentiate into neurons after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, approximately 90% of the NPCs differentiate into neurons after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, approximately 91% of the NPCs differentiate into neurons after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, approximately 92% of the NPCs differentiate into neurons after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, approximately 93% of the NPCs differentiate into neurons after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, approximately 94% of the NPCs differentiate into neurons after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, approximately 95% of the NPCs differentiate into neurons after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, 5%-15% of the NPCs differentiate into glial cells after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, 5%-10% of the NPCs differentiate into glial cells after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, 10%-15% of the NPCs differentiate into glial cells after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, approximately 5% of the NPCs differentiate into glial cells after transplantation to a site of nerve injury or disease.In another instance, in the presence of the compositions disclosed herein, approximately 10% of NPCs differentiated into glial cells after transplantation to a site of nerve injury or disease. In another instance, in the presence of the compositions disclosed herein, approximately 15% of NPCs differentiated into glial cells after transplantation to a site of nerve injury or disease.

[0105] In one instance, in the presence of the compositions disclosed herein, NPCs differentiated into neurons and / or glial cells approximately one to six months after transplantation to a site of nerve injury or disease. In one instance, in the presence of the compositions disclosed herein, NPCs differentiated into neurons and / or glial cells approximately 30 days after transplantation to a site of nerve injury or disease. In one instance, in the presence of the compositions disclosed herein, NPCs differentiated into neurons and / or glial cells approximately 40 days after transplantation to a site of nerve injury or disease. In one instance, in the presence of the compositions disclosed herein, NPCs differentiated into neurons and / or glial cells approximately 50 days after transplantation to a site of nerve injury or disease. In one instance, in the presence of the compositions disclosed herein, NPCs differentiated into neurons and / or glial cells approximately 60 days after transplantation to a site of nerve injury or disease. In one instance, in the presence of the compositions disclosed herein, NPCs differentiated into neurons and / or glial cells approximately three months after transplantation to a site of nerve injury or disease. In one instance, in the presence of the compositions disclosed herein, NPCs differentiated into neurons and / or glial cells approximately four months after transplantation to a site of nerve injury or disease. In one instance, in the presence of the compositions disclosed herein, NPCs differentiated into neurons and / or glial cells approximately five months after transplantation to a site of nerve injury or disease. In one instance, in the presence of the compositions disclosed herein, NPCs differentiated into neurons and / or glial cells approximately six months after transplantation to a site of nerve injury or disease. In one instance, in the presence of the compositions disclosed herein, the axons of the neurons differentiated from NPCs projected into the CNS tissues, such as the brainstem, spinal cord, and contralateral cortex, to reconnect the ischemic cortex to the rest of the brain approximately six months after transplantation.

[0106] The compositions disclosed herein that comprise a gel-forming molecule and a CCR5 antagonist support the survival and differentiation of NPCs transplanted to a site of nerve injury or disease. In one example, the gel-forming molecule is fibrinogen. The fibrinogen molecule is a 340-kDa homodimeric glycoprotein composed of 2Aα, 2Bβ, and 2γ polypeptide chains linked by 29 disulfide bonds. In the presence of thrombin, thrombin cleaves fibrinopeptides to form fibrin monomers. These monomers then polymerize in a semi-staggered arrangement to form fibrinogen fibrils and ultimately a fibrin network, an insoluble gel. In addition, calcium reduces the time required to form fibrin from fibrinogen by significantly accelerating the stage of fibrin monomer polymerization. Advantageously, once fibrinogen forms a gel in the presence of thrombin or calcium, the gel provides a scaffold for the stable transplantation of NPCs at the site of nerve injury or disease. In addition, fibrinogen is neurotrophic and it supports the growth of transplanted NPCs. Further, the gel formed by fibrinogen prevents the rapid dilution and / or degradation of maraviroc and supports the survival of transplanted NPCs.

[0107] In one example, the concentration of fibrinogen in the compositions disclosed herein is from 5 mg / mL to 30 mg / mL. In another example, the concentration of fibrinogen in the compositions disclosed herein is about 5 mg / mL. In another example, the concentration of fibrinogen in the compositions disclosed herein is about 9 mg / mL. In another example, the concentration of fibrinogen in the compositions disclosed herein is about 10 mg / mL. In another example, the concentration of fibrinogen in the compositions disclosed herein is about 15 mg / mL. In another example, the concentration of fibrinogen in the compositions disclosed herein is about 20 mg / mL. In another example, the concentration of fibrinogen in the compositions disclosed herein is about 25 mg / mL. In another example, the concentration of fibrinogen in the compositions disclosed herein is about 30 mg / mL.

[0108] 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).

[0109] The compositions disclosed herein comprise CCR5 antagonists. CCR5 is an inflammatory chemokine receptor. In the immune system, CCR5 is mainly expressed on T cells, macrophages, dendritic cells, and eosinophils. Effector CCR5 + T cells are directed by chemokines produced in local tissues and innate immune cells activated at that site to the sites of infection and inflammation, leading to 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 1 (MIP), also known as MIP1-α and MIP1-β, respectively. CCR5 is expressed on neural stem cells (NSCs) or neural progenitor cells, but not on mature neurons. As a chemokine receptor, in the case of nerve injury or disease, it helps to recruit CCR5 + cells together with CCR5 ligands (such as chemokines caused by inflammation) to the sites of nerve injury or disease. Therefore, NSCs or neural progenitor cells tend to form clusters or neural rosettes at the sites of nerve injury or disease, which keeps the NSCs or neural progenitor cells in an immature stem cell or progenitor cell state. If CCR5 signaling is blocked, less aggregation or a more uniform distribution will occur, and thus the cells will differentiate or mature.

[0110] As used herein, the term "CCR5 antagonist" refers to a molecule that inhibits or 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 or attenuates or reduces the biological activity of CCR5 by interfering with the interaction of CCR5 with another molecule such as its ligand CCL3 / 4 / 5. In one example, a CCR5 antagonist is a molecule that inhibits or attenuates or reduces the biological activity of CCR5 by acting on components of the biological pathway in which CCR5 is involved. In another example, a CCR5 antagonist is a molecule that reduces the gene expression encoding CCR5 and thus reduces the protein level of CCR5. A CCR5 antagonist can be a molecule selected from the group consisting of small molecules, nucleic acids, antibodies, anticalins, carbohydrates, and any other compound or composition that inhibits or attenuates or reduces the activity of CCR5 by directly interacting with CCR5 or by acting on components of the biological pathway in which CCR5 is involved or by reducing the protein expression level of CCR5.

[0111] In one instance, the CCR5 antagonist is a small molecule. In another instance, the CCR5 antagonist is maraviroc. Maraviroc (trademarked outside the United States as Selzentry or Celsentri) is a chemokine receptor antagonist drug developed by Pfizer Pharmaceuticals that combats HIV by interfering with the interaction between HIV and CCR5. It was approved for use by the FDA in August 2007. In one instance, maraviroc blocks the activation of CCR5, which is expressed on transplanted NPCs by inflammatory cytokines such as CCL released by inflammatory cells infiltrating the site of nerve injury or disease, reducing apoptosis and promoting the differentiation / maturation of transplanted NPCs. Thus, maraviroc can effectively reduce inflammatory damage in the adverse inflammatory environment at the site of nerve injury or disease (such as the infarct core).

[0112] In one instance, the concentration of maraviroc in the compositions disclosed herein is 3 mg / mL - 50 mg / mL. In another instance, the concentration of maraviroc in the compositions disclosed herein is approximately 3 mg / mL. In another instance, the concentration of maraviroc in the compositions disclosed herein is approximately 5 mg / mL. In another instance, the concentration of maraviroc in the compositions disclosed herein is approximately 10 mg / mL. In another instance, the concentration of maraviroc in the compositions disclosed herein is approximately 15 mg / mL. In another instance, the concentration of maraviroc in the compositions disclosed herein is approximately 20 mg / mL. In another instance, the concentration of maraviroc in the compositions disclosed herein is approximately 25 mg / mL. In another instance, the concentration of maraviroc in the compositions disclosed herein is approximately 30 mg / mL. In another instance, the concentration of maraviroc in the compositions disclosed herein is approximately 35 mg / mL. In another instance, the concentration of maraviroc in the compositions disclosed herein is approximately 40 mg / mL. In another instance, the concentration of maraviroc in the compositions disclosed herein is approximately 45 mg / mL. In another instance, the concentration of maraviroc in the compositions disclosed herein is approximately 50 mg / mL.

[0113] In another instance, 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.

[0114] In another instance, the CCR5 antagonist is a nucleic acid. In another instance, the CCR5 antagonist is a small interfering RNA (siRNA). Small interfering RNAs (siRNAs) are typically double-stranded RNA molecules that are 20-25 nucleotides in length. When transfected into cells, siRNAs transiently inhibit the target mRNA until they are also degraded within the cell. In another instance, the CCR5 antagonist is a short hairpin RNA (shRNA). Short hairpin RNAs (shRNAs) are RNA sequences that are typically about 80 base pairs in length and include an internal hybridization region that produces a hairpin structure. shRNA molecules are processed intracellularly to form siRNAs, which in turn knockdown gene expression. In another instance, the CCR5 antagonist is a microRNA (miRNA). miRNAs are small non-coding RNAs that are on average 22 nucleotides in length. miRNAs are partially complementary to one or more messenger RNA (mRNA) molecules, and they can downregulate gene expression in a variety of ways, including translational repression, mRNA cleavage, and deadenylation.

[0115] In one instance, the concentration of siRNA in the compositions disclosed herein is 1x10 6 -1x10 8 U / mL. In another instance, the concentration of shRNA in the compositions disclosed herein is about 1x10 6 -1x10 8 U / mL. In another instance, the concentration of miRNA in the compositions disclosed herein is about 1x10 6 -1x10 8 U / mL. In another instance, the concentration of siRNA or shRNA or miRNA in the compositions disclosed herein is about 1x10 6 U / mL. In another instance, the concentration of siRNA or shRNA or miRNA in the compositions disclosed herein is about 5x10 6 U / mL. In another instance, the concentration of siRNA or shRNA or miRNA in the compositions disclosed herein is about 1x10 7 U / mL. In another instance, the concentration of siRNA or shRNA or miRNA in the compositions disclosed herein is about 5x10 7 U / mL. In another instance, the concentration of siRNA or shRNA or miRNA in the compositions disclosed herein is about 1x10 8 U / mL.

[0116] In one example, a 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 generally have high transfection efficiency. In another example, a nucleic acid-based CCR5 antagonist is delivered to NPCs using a non-viral vector. In one example, the non-viral vector is an inorganic material-based vector selected from the group consisting of gold nanoparticles (AuNPs), mesoporous silica, graphene oxide, and Fe3O4-mediated nanoparticles (NPs). In another example, the non-viral vector is a lipid-based nanocarrier selected from the group consisting of cationic lipids such as Lipofectamine and neutral lipids such as cholesterol, dioleoylphosphatidylcholine (DOPC), and dioleoylphosphatidylethanolamine (DOPE). In another example, the non-viral vector is a polymeric vector selected from the group consisting of polyethylenimine (PEI), poly(lactide-co-glycolide) (PLGA), chitosan, and β-cyclodextrin. In another example, the non-viral vector is a dendrimer-based vector such as a PAMAM dendrimer.

[0117] In another example, the CCR5 antagonist is an antibody. In another example, the CCR5 antagonist is the antibody PRO140 (Leronlimab).

[0118] As used herein, the term "neural progenitor cell (NPC)" refers to a heterogeneous population of cells consisting of all undifferentiated progeny of neural stem cells (NSCs), and thus includes NSCs and neural progenitor cells. The term "neural progenitor cell (NPC)" is commonly used to collectively refer to the heterogeneous population of NSCs and neural progenitor cells.

[0119] As used herein, the term "neural stem cell (NSC)" refers to a pluripotent cell of the central nervous system (CNS, consisting of the brain and spinal cord) that is capable of self-renewing and proliferating indefinitely and giving rise to daughter cells that ultimately differentiate into many (if not all) of the glial cell and neuronal cell types that arise in the CNS, such as neurons or glial cells, including astrocytes and oligodendrocytes. The non-stem cell progeny of NSCs are referred to as neural progenitor cells.

[0120] 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 pluripotent. A notable feature of neural progenitor cells is that, unlike stem cells, they have limited proliferative capacity and do not exhibit self-renewal.

[0121] In one instance, NPCs are generated in vitro by differentiating embryonic stem cells (ESCs). In another instance, NPCs are generated in vitro by differentiating induced pluripotent stem cells (iPSCs). iPSCs are derived from somatic cells, most commonly fibroblasts or blood cells, and are reprogrammed to an embryo-like pluripotent state.

[0122] In one instance, the NPCs are embryonic NPCs isolated from the CNS of a developing embryo. During mammalian CNS development, NPCs from the neural tube give rise to a pool of pluripotent and more restricted neural progenitor cells that then proliferate, migrate, and further differentiate into neurons and glial cells. During embryogenesis, NPCs are derived from the neuroectoderm and can first be detected during neural plate and neural tube formation. 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 unique spatial properties and differentiation potential.

[0123] In another instance, the NPCs are adult NPCs isolated from the CNS of a mature adult. In another instance, 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, α2 regions and the "hypothalamic proliferative zone" located adjacent to the median eminence).

[0124] NPCs can be grafted or transplanted to the site of nerve injury or disease together with the compositions disclosed herein that support the survival and differentiation of transplanted NPCs. As used herein, the term "site of nerve injury or disease" refers to a site resulting from a nerve injury or disease characterized by inflammation, 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, nerve cell injury induced by hypoxia such as in cardiac arrest or neonatal distress, cancer-related neurological conditions, and neurodegenerative diseases. In one example, the site of nerve injury or disease is caused by a stroke, particularly a cerebral stroke. A cerebral stroke refers to the sudden and permanent death of brain cells when blood flow is blocked and oxygen cannot be delivered to the brain. In one example, the cerebral stroke is an ischemic stroke. Ischemic strokes most commonly occur when blood flow is blocked due to blood clotting (referred to as arterial "thrombosis") or detached blood clots lodged in an artery (referred to as an "embolic stroke"). In another example, the cerebral stroke is a hemorrhagic stroke. A hemorrhagic stroke is caused by a rupture of the arterial wall and leakage of blood into the surrounding brain. Like ischemic strokes, hemorrhagic strokes are a cause of tissue death by depriving the brain of blood and oxygen and result in many neurological disabilities (motor, speech) as well as functional disabilities. In another example, the site of nerve injury or disease is caused by a traumatic brain injury. Traumatic brain injuries are typically caused by a violent blow or jolt to the head or body. Objects passing through the brain tissue, such as a bullet or skull fragment, can also cause a traumatic brain injury. Traumatic brain injuries can result in contusions, tissue tears, bleeding, and other physical damage to the brain. These injuries can lead to long-term complications or death. In one example, the traumatic brain injury is a closed head injury. A closed head injury occurs when the brain is injured non-penetratingly without a skull fracture, which is caused by the rapid forward or backward movement and concussion of the brain within the skull, resulting in contusions and tears 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 the skull is fractured, such as when a bullet penetrates the brain. In another example, the site of nerve injury or disease is caused by a spinal cord injury. A spinal cord injury refers to an injury to any part of the spinal cord or the nerves at the end of the spinal canal (cauda equina), which typically results in permanent changes in strength, sensation, and other body functions below the site of injury. In another example, the site of nerve injury or disease is caused by multiple sclerosis (MS). In MS, the immune system attacks the protective sheath (myelin) covering nerve fibers and can cause permanent damage or deterioration of the nerves. In another example, the site of nerve injury or disease is caused by Alzheimer's disease, which is characterized by damaged nerve cells. In another example, the site of nerve injury or disease is caused by Parkinson's disease. In Parkinson's disease, the nerve cells in the basal ganglia (the area of the brain that controls movement) are damaged and / or die.In another instance, the site of nerve injury or disease is caused by Huntington's disease, which causes movement, cognitive, and psychiatric disorders with a wide range of signs and symptoms. In another instance, the site of nerve injury or disease is caused by amyotrophic lateral sclerosis (ALS). ALS affects the nerve cells (motor neurons) that control voluntary muscle movements such as walking and speaking. ALS causes the gradual degeneration and then death of motor neurons.

[0125] In one instance, the site of nerve injury or disease is in the brain. In another instance, the site of nerve injury or disease is in the spinal cord. In another instance, the site of nerve injury or disease is in the telencephalon. In another instance, the site of nerve injury or disease is in the cerebellum. In another instance, the site of nerve injury or disease is in the brainstem. In another instance, the site of nerve injury or disease is in the frontal lobe of the telencephalon. In another instance, the site of nerve injury or disease is in the parietal lobe of the telencephalon. In another instance, the site of nerve injury or disease is in the occipital lobe of the telencephalon. In another instance, the site of nerve injury or disease is in the temporal lobe of the telencephalon. In another instance, the site of nerve injury or disease is in the midbrain of the brainstem. In another instance, the site of nerve injury or disease is in the pons of the brainstem. In another instance, the site of nerve injury or disease is in the medulla. In another instance, the site of nerve injury or disease is in two or more of the sites described above. In another instance, the site of nerve injury or disease is in a brain region selected from the group consisting of the pituitary gland, hypothalamus, amygdala, hippocampus, pineal gland, ventricles, and cerebrospinal fluid. In another instance, the site of nerve injury or disease has an impaired 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 instance, the site of nerve injury or disease has an impaired 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 instance, the site of nerve injury or disease is focal (confined to one area of the brain). In another instance, the site of nerve injury or disease is diffuse (occurring in more than one area of the brain).

[0126] In animals with a nerve injury or disease (such as an ischemic stroke) disclosed herein, the site of nerve injury or disease has dead nerve cells, or impaired blood vessels, or both. In one instance, the ischemic / infarct site forms a cavity isolated by a glial scar and filled with inflammatory cells and secretions. This inflammatory environment is not conducive to transplanted NPCs, such that few transplanted NPCs survive. The compositions disclosed herein support the survival and differentiation of transplanted NPCs at the site of nerve injury or disease disclosed herein.

[0127] In one instance, the compositions disclosed herein that include a gel-forming molecule and a CCR5 antagonist further include calcium. In another instance, the composition that includes fibrinogen and maraviroc further includes CaCl2. Advantageously, calcium reduces the time required to form fibrin from fibrinogen by significantly accelerating the stages of fibrin monomer polymerization and gel formation. The gel effectively holds the transplanted NPCs together and prevents the rapid dilution and / or degradation of maraviroc, supporting the survival of the transplanted NPCs. In one instance, the concentration of CaCl2 in the compositions disclosed herein is 1 - 5 mM. In a particular instance, the concentration of CaCl2 in the compositions disclosed herein is 2.5 mM.

[0128] In another instance, the compositions disclosed herein that include a gel-forming molecule and a CCR5 antagonist do not include calcium. After the mixture of NPCs and the compositions disclosed herein is administered to the site of nerve injury or disease, the calcium present in the body fluid will promote gel formation.

[0129] In another instance, the compositions disclosed herein that include a gel-forming molecule and a CCR5 antagonist further include thrombin. Thrombin mediates proteolytic cleavage and removal of the N-terminal fibrinopeptides from the Aα and Bβ chains of fibrinogen and results in the formation of fibrin (gel). In one instance, the concentration of thrombin in the compositions disclosed herein is 10 U / mL - 500 U / mL. In another instance, the concentration of thrombin in the compositions disclosed herein is approximately 10 U / mL. In another instance, the concentration of thrombin in the compositions disclosed herein is approximately 50 U / mL. In another instance, the concentration of thrombin in the compositions disclosed herein is approximately 100 U / mL. In another instance, the concentration of thrombin in the compositions disclosed herein is approximately 200 U / mL. In another instance, the concentration of thrombin in the compositions disclosed herein is approximately 300 U / mL. In another instance, the concentration of thrombin in the compositions disclosed herein is approximately 400 U / mL. In another instance, the concentration of thrombin in the compositions disclosed herein is approximately 500 U / mL.

[0130] In another instance, the compositions disclosed herein that include a gel-forming molecule and a CCR5 antagonist do not include thrombin. After the mixture of NPCs and the compositions disclosed herein is administered to the site of nerve injury or disease, the thrombin present in the body fluid will promote gel formation.

[0131] 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 (BMP4). Depending on the nature of the NPCs, the number of NPCs to be transplanted, and the size of the lesion to be filled, the growth factor can be used in the compositions disclosed herein. In one example, if the site of nerve injury or disease (such as a lesion) is large and more cells need to be divided to fill it, the growth factor disclosed herein is included in the composition. In another example, the concentration of brain-derived neurotrophic factor (BDNF) in the composition is 1 ng / mL - 100 ng / mL. In another example, the concentration of nerve growth factor (DNF) in the composition is 1 ng / mL - 100 ng / mL. In another example, the concentration of neurotrophin in the composition is 1 ng / mL - 50 ng / mL. In another example, the concentration of platelet-derived growth factor (PDGF) in the composition is 1 ng / mL - 100 ng / mL. In another example, the concentration of glial cell line-derived neurotrophic factor (GDNF) in the composition is 1 ng / mL - 500 ng / mL. In another example, the concentration of insulin-like growth factor-1 (IGF-1) in the composition is 1 ng / mL - 50 ng / mL. In another example, the concentration of insulin-like growth factor-2 (IGF-2) in the composition is 1 ng / mL - 50 ng / mL. In another example, the concentration of fibroblast growth factor (FGF) in the composition is 1 ng / mL - 200 ng / mL. In another example, the concentration of vascular endothelial growth factor (VEGF) in the composition is 10 ng / mL - 500 ng / mL. In another example, the concentration of bone morphogenetic protein (BMP4) in the composition is 5 ng / mL - 500 ng / mL. Advantageously, the concentration of the growth factor in the compositions disclosed herein is similar to its physiological concentration. This avoids the problem in the art of routinely using microgram levels of growth factors with fibrinogen to support transplanted cells at the site of injury, where the conventionally used growth factors have a concentration that is more than 1000 times the physiological concentration. At such high concentrations, these growth factors can promote the proliferation of transplanted NPCs, forming "occupying" tissue similar to tumors in the spinal cord, which may cause secondary injury by compressing intact areas. Using growth factors at high concentrations (more than 1000 times the physiological concentration) also inhibits the differentiation of NPCs. Thus, the transplanted NPCs in conventional studies remain in the neural precursor state for months.In contrast, the concentrations of growth factors used herein prevent the occurrence of a situation in which transplanted NPCs remain in a proliferative state resulting in overgrowth into "place-occupying" tissue.

[0132] In another example, the compositions disclosed herein do not include growth factors selected from the group consisting of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophic factor, platelet-derived growth factor (PDGF), glial cell-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 (BMP4). This prevents the overgrowth of transplanted NPC in the case of relatively small lesions.

[0133] In another aspect, the present disclosure relates to a method for treating neural injury or disease in a subject, comprising (a) mixing NPCs with a composition disclosed herein; and (b) administering the mixture of NPCs and the composition to the site of neural injury or disease in the subject, thereby supporting the survival and differentiation of NPCs.

[0134] In another aspect, the present disclosure relates to the use of a mixture of NPCs and a composition disclosed herein in the preparation of a medicament for treating neural injury or disease in a subject, wherein the mixture is administered to the site of neural injury or disease in the subject, thereby supporting the survival and differentiation of NPCs.

[0135] As used herein, the term "treatment" and grammatical variations of the term refer to administering a mixture of NPC and the compositions 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 in any manner, prevent the development of a disease, or otherwise prevent, hinder, delay or reverse a disease or other adverse symptoms.

[0136] As disclosed herein, the term "neural progenitor cell (NPC)" is used to collectively refer to a mixed population of NSCs and neural progenitor 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 somatic cells, most commonly fibroblasts or blood cells, and are reprogrammed 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 from the neural tube give rise to a pool of pluripotent 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 during neural plate and neural tube formation. 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 unique spatial properties and differentiation potential. In another example, 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 hippocampal dentate gyrus, the subventricular zone surrounding the lateral ventricle, and the hypothalamus (specifically, in the dorsal α1, α2 regions and the "hypothalamic proliferative zone" located adjacent to the median eminence).

[0137] In one example, the nerve 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, hypoxic-induced nerve cell injury such as in cardiac arrest or neonatal distress, cancer-related neurological conditions, and neurodegenerative diseases. In one example, the nerve injury or disease is a stroke, particularly a cerebral stroke. A cerebral stroke refers to the sudden and permanent death of brain cells when blood flow is blocked and oxygen cannot be delivered to the brain. In one example, the cerebral stroke is an ischemic stroke. Ischemic strokes most commonly occur when blood flow is blocked due to blood clotting (referred to as arterial "thrombosis") or detached blood clots lodged in the artery (referred to as "embolic stroke"). In another example, the cerebral stroke is a hemorrhagic stroke. A hemorrhagic stroke is caused by a rupture of the arterial wall and leakage of blood into the surrounding brain. Like ischemic strokes, hemorrhagic strokes are a cause of tissue death by depriving the brain of blood and oxygen and result in many neurological disabilities (motor, language) as well as functional disabilities. In another example, the nerve injury or disease is a traumatic brain injury. Traumatic brain injuries are typically caused by a violent blow or jolt to the head or body. Objects passing through brain tissue, such as a bullet or skull fragment, can also cause a traumatic brain injury. Traumatic brain injuries can result in contusions, tissue tearing, bleeding, and other physical damage to the brain. These injuries can lead to long-term complications or death. In one example, the traumatic brain injury is a closed head injury. A closed head injury occurs when the brain is injured non-penetratingly without a skull fracture, which is caused by the rapid forward or backward movement and concussion of the brain within the skull, resulting in contusions and tearing 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 the skull is fractured, such as when a bullet penetrates the brain. In another example, the nerve injury or disease is a spinal cord injury. A spinal cord injury refers to an injury to any part of the spinal cord or the nerves at the end of the spinal canal (cauda equina), which typically results in permanent changes in strength, sensation, and other body functions below the site of injury. In another example, the nerve injury or disease is multiple sclerosis (MS). In MS, the immune system attacks the protective sheath (myelin) covering nerve fibers and can cause permanent damage or deterioration of the nerves. In another example, the nerve injury or disease is Alzheimer's disease, which is characterized by damaged nerve cells. In another example, the nerve injury 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 injury or disease is Huntington's disease, which causes movement, cognitive, and psychiatric disorders with a wide range of signs and symptoms. In another example, the nerve injury or disease is amyotrophic lateral sclerosis (ALS).ALS affects the nerve cells (motor neurons) that control voluntary muscle movements such as walking and speaking. ALS causes the motor neurons to gradually degenerate and then die.

[0138] The terms "subject", "host", and "patient" are used interchangeably. As used herein, the subject is preferably a mammal, such as a non - primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human), and most preferably human.

[0139] In one instance, the number of NPCs mixed with and administered to the site of nerve injury or disease disclosed herein is 50,000 - 500,000. In another instance, the number of NPCs mixed with and administered to the site of nerve injury or disease disclosed herein is 50,000 - 100,000. In another instance, the number of NPCs mixed with and administered to the site of nerve injury or disease disclosed herein is 100,000 - 200,000. In another instance, the number of NPCs mixed with and administered to the site of nerve injury or disease disclosed herein is 200,000 - 300,000. In another instance, the number of NPCs mixed with and administered to the site of nerve injury or disease disclosed herein is 300,000 - 400,000. In another instance, the number of NPCs mixed with and administered to the site of nerve injury or disease disclosed herein is 400,000 - 500,000. In another instance, the number of NPCs mixed with and administered to the site of nerve injury or disease disclosed herein is about 50,000. In another instance, the number of NPCs mixed with and administered to the site of nerve injury or disease disclosed herein is about 100,000. In another instance, the number of NPCs mixed with and administered to the site of nerve injury or disease disclosed herein is about 200,000. In another instance, the number of NPCs mixed with and administered to the site of nerve injury or disease disclosed herein is about 300,000. In another instance, the number of NPCs mixed with and administered to the site of nerve injury or disease disclosed herein is about 400,000. In another instance, the number of NPCs mixed with and administered to the site of nerve injury or disease disclosed herein is about 500,000. In another instance, the number of NPCs mixed with and administered to the site of nerve injury or disease disclosed herein exceeds 500,000.

[0140] In one example, NPCs are mixed with the compositions disclosed herein that include a gel-forming molecule, a CCR5 antagonist, and thrombin, and the mixture is administered to a site of nerve injury or disease after mixing. In another example, NPCs are mixed with the compositions disclosed herein that include a gel-forming molecule and a CCR5 antagonist, and the mixture is administered to a site of nerve injury or disease, and then thrombin is administered to the site of nerve injury or disease. Thrombin can be added separately and added after administering the mixture of NPCs and the compositions disclosed herein that include a gel-forming molecule and a CCR5 antagonist to avoid premature gel formation prior to transplantation.

[0141] As used herein, the term "administer" and grammatical variants of the term refer to transplanting a mixture of NPCs and the compositions disclosed herein to a site of nerve injury or disease in a subject disclosed herein. In one example, the mixture of NPCs and the compositions disclosed herein is administered by stereotactic injection, which allows direct injection of the NPCs to the site of nerve injury or disease disclosed herein.

[0142] Cell therapy for nerve injury and diseases 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, hypoxic-induced nerve cell injury such as in cardiac arrest or neonatal distress, cancer-related neuropathies, and neurodegenerative diseases requires coordinated and dynamic regulation of transplanted NPCs, including maximal survival, an appropriate degree of proliferation, neuron vs. 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 the NPCs are transplanted to a site of nerve injury or disease. Since it was found that CCR5 is highly expressed on NPCs, CCR5 antagonists promote NPC survival by blocking CCR5 signaling. In addition, the gel-forming molecule forms a gel that holds the transplanted NPCs together and prevents the transplanted NPCs from "swimming" in the lesion cyst or cavity. In the presence of fibrinogen and maraviroc, the transplanted NPCs are evenly distributed without aggregation and differentiate into mature neurons at about 30 days post-transplantation.

[0143] In another aspect, the invention relates to a kit for supporting the survival and differentiation of NPCs transplanted to a site of nerve injury or disease, the kit comprising: (a) the compositions disclosed herein; (b) artificial cerebrospinal fluid (a-CSF); (c) CaCl2; and (d) thrombin.

[0144] In one example, the kit further includes a carrier, diluent, and adjuvant for administering the mixture of NPCs and the compositions disclosed herein. The carrier, diluent, and adjuvant must be pharmaceutically "acceptable" in terms of compatibility with the other components of the composition and harmless to its recipient.

[0145] Examples of pharmaceutically acceptable carriers or diluents are softened or distilled water; saline solutions; vegetable-based 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 hydroxypropylmethylcellulose; lower alkanols such as ethanol or isopropanol; lower aralkanol; lower polyalkylene glycols or lower alkylene glycols such as polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butanediol or glycerol; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrrolidone; agar; tragacanth or gum arabic, and petrolatum. Generally, one or more carriers will comprise from 10% to 99.9% by weight of the composition.

[0146] Unless the context clearly indicates otherwise, as used in this application, the singular forms "a", "an" and "the" include plural referents. For example, the term "a primer" includes multiple primers, including mixtures and combinations thereof.

[0147] As used herein, the term "comprising" means "including". Variations of the word "comprising", such as "comprise" and "comprises", have corresponding different meanings. Thus, for example, a composition "comprising" X can consist solely of X or can include one or more additional unrecited components.

[0148] 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 specified value means + / - 5% of the specified value, or + / - 4% of the specified value, or + / - 3% of the specified value, or + / - 2% of the specified value, or + / - 1% of the specified value, or + / - 0.5% of the specified value.

[0149] 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 disclosed range. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as the individual numerical values within that range. For example, a description of a range from 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as the individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies to ranges of any width.

[0150] The invention described illustratively herein may be practiced appropriately without any one or more of the elements, one or more limitations not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc. should be read broadly and without limitation. In addition, the terms and expressions used herein are used as terms of description and not of limitation, and there is no intention to exclude any equivalents of the features shown and described or portions thereof, but it should be recognized that various modifications are possible within the scope of the invention claimed. Accordingly, it should be understood that although the invention has been specifically disclosed by preferred embodiments and optional features, those skilled in the art may make modifications and variations to the invention disclosed herein, and such modifications and variations are considered to be within the scope of the invention.

[0151] The invention has been described herein broadly and generically. Each narrower genus and sub-genus group falling within the general disclosure also forms part of the invention. This includes the general description of the invention with the proviso or negative limitation of removing any subject matter from that genus, regardless of whether the material removed is specifically recited herein.

[0152] Unless otherwise noted, 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.

[0153] Other embodiments are in the appended claims and the non-limiting examples below.

[0154] Examples

[0155] Non-limiting examples of the present disclosure will be described in further detail by reference to specific examples, which should not be construed as limiting the scope of the present disclosure in any way.

[0156] Example 1 - Method

[0157] Cell culture

[0158] Human embryonic stem cells (ESCs, eGFP H9 hESC line) were cultured in feeder-free mTeSR medium containing 1× mTeSR supplement, 1× non-essential amino acids (NEAA), and 1× Glutamax in Matrigel-coated 6-well plates. The ESCs were fed daily and passaged twice a week.

[0159] The following protocol was used for the induction of forebrain glutamate NPCs. Briefly, H9 hESCs or eGFP H9 hESCs were cultured in Matrigel / vitronectin-coated 6-well plates for one week. On day 0, the ESC colonies were gently dissociated with a 1 mL pipette to form cell aggregates. The cell aggregates were cultured in a flask with neural induction medium (NIM) composed of DMEM / F12, 1× N2 supplement, 1× NEAA, 2 μM SB431542, and 2 μM DMH-1 for 7 days. In the presence of NIM containing 5% FBS, the cell aggregates were allowed to adhere to the 6-well plates for 6 hours and then fresh NIM was replaced. The aggregates were fed with NIM until neurospheres formed on day 16. The neurospheres were gently dissociated with a 1 mL pipette and suspended in a flask containing NIM for 7 days. Then, starting from day 23, the NIM was changed every four days. Neural progenitor cells (NPCs) were maintained in NIM until transplantation or immunostaining. On day 49, the NPCs were digested with TrypLE for 3 min to make single cells. After an additional day of culture in NIM supplemented with 1× B27 and 100 nM compound E, the NPCs were collected for transplantation in the ischemic stroke model. The B-27 supplement is a defined but complex mixture of antioxidant enzymes, proteins, vitamins, and fatty acids, which are combined in an optimized ratio to support neuronal survival in culture. Compound E is a γ-secretase inhibitor. For immunostaining, the NPCs were seeded on coverslips and stained after one week of culture.

[0160] To test the effects of CCR5 activation on NPCs and neurons, the cells were seeded on Matrigel-coated coverslips or 6-well plates and treated with an excess (300 ng / mL) of CCR5 ligand. CCL3, CCL4, CCL5, and their combination (CCL3 / 4 / 5) were added to the NIM every other day, respectively. After four days of treatment, the NPCs or neurons were collected for staining or immunoblotting.

[0161] Lentivirus production

[0162] Obtained the human CCR5 29mer shRNA plasmid (pRS_hU6_CCR5shRNA_SV40_Puro) and the scrambled 29-mer shRNA cassette in the pRS vector from OriGene (Catalog No.: TR314126, Catalog No.: TR30012). Lentiviral shRNA was produced in the HEK 293FT cell line by transfection of the packaging plasmid and the backbone plasmid. HEK293FT cells were cultured in DMEM containing 10% FBS. The supernatant was collected after 3 days of culture. The viral particles were concentrated by ultracentrifugation at 25,000 rpm for 2.5 h at 4°C. The viral particles were resuspended in DMEM.

[0163] Transduce shRNA

[0164] Seed 1x10 ^5 NPCs on the coverslips in each well of a 24-well plate for 2 days until 50% confluent and transduce them in a humidified 5% CO2 incubator at 37°C. Infect the NPCs overnight at 37°C with lentiviral shRNA (MOI of 20). Remove the medium containing the lentiviral particles from the wells and replace it with 500 μL of fresh pre-warmed NIM. At 5 days post-transduction, collect the infected NPCs for immunostaining or western blotting.

[0165] Prepare the mixture / composition

[0166] Prepare stock solutions of 30 mg / mL fibrinogen, 50 mg / mL maraviroc, and 250 mM CaCl2 (100×) in the following manner: Dissolve fibrinogen (F3879, Sigma) in α-CSF for 1 h at room temperature. Dissolve maraviroc in dimethyl sulfoxide (DMSO). Dissolve CaCl2 (Sigma) in deionized (DI) water. All solutions were filter-sterilized and stored at -20°C for later use. The mixture / composition was made of 50 mg / mL maraviroc and 30 mg / mL fibrinogen in a 1:9 volume ratio with 2.5 mM CaCl2.

[0167] Release profile of maraviroc in the mixture gel

[0168] By scanning in the UV range from 2000 nm to 400 nm, the maximum absorption wavelength (λ max ) of maraviroc in phosphate buffer (pH 7.4) was found to be 210 nm. Prepare a standard drug solution of maraviroc by dissolving 50 mg of pure maraviroc in phosphate buffer 7.4 and transfer it to a 5 mL volumetric flask to obtain a 10 mg / mL stock solution and the resulting maraviroc. The solution was used as the working standard solution from which solutions of the required concentrations were prepared. Using an appropriate blank, at λ maxMeasure the absorbance at 210 nm for final concentrations of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 mg / mL. Induce the gelation of 200 μL fibrinogen (9 mg / mL) in vitro with thrombin (50 U) and incubate the gel containing 1 mg maraviroc in 1 mL PBS (pH = 7.4) at 37 °C. For the free drug group, dissolve 5 mg maraviroc directly in 1 mL PBS. Take 50 μL of the solution and measure the absorbance with a microplate absorbance spectrophotometer (Bio-RAD, xMark TM ) Test the absorbance. Calculate the final concentration of maraviroc in PBS according to the calibration curve.

[0169] Stroke Model and Cell Transplantation

[0170] All animal studies were conducted in accordance with the requirements of the Institutional Animal Care and Use Committee of Duke-NUS Medical School. Induce ischemic stroke in adult (10 - 12 weeks) male SCID mice by photochemical embolization. Briefly, inject Rose Bengal intravenously at a dose of 0.1 mg / g per mouse. Expose the mouse skull under 2% isoflurane anesthesia. The right motor cortex (anterior-posterior [AP] = +2 mm, lateral [L] = +1 mm) was irradiated with cold light of 2.5 mm in diameter through the intact skull for 15 min. Randomly divide the animals and transplant forebrain glutamatergic precursors. In the presence or absence of maraviroc (5 mg / mL) or fibrinogen (10 mg / mL), resuspend 50,000 cells in 1 μL of artificial cerebrospinal fluid (a-CSF) and inject them into the lesion site (from the dura mater [AP] = +2 mm, [L] = +1 mm, vertical [V] = -1.5 mm).

[0171] Tissue Preparation and Immunohistochemistry

[0172] Euthanize the animals with a lethal dose of pentobarbital (250 mg / kg) and immediately perfuse with PBS, then perfuse with 4% cold paraformaldehyde (PFA). Fix the brain samples in cold PFA for 2 hours and immerse them in 30% sucrose at 4 °C for about 4 days until they sink. Collect serial coronal (from 1.54 mm anterior to the bregma to -0.22 mm) sections at a thickness of 40 - μm on a cryostat microtome and store them at -20 °C. For immunostaining, incubate the sections with a blocking solution containing 10% normal donkey serum and 0.2% Triton-100 at room temperature for 1 h. Then incubate the sections with the primary antibody overnight at 4 °C.

[0173]

[0174]

[0175]

[0176]

[0177] Subsequently, the sections were washed and incubated with the corresponding secondary antibody for 1 h at room temperature. The immunolabeled sections were fixed with Hoechst via Fluoromount-G. For TUNEL staining, the cells on the coverslips were fixed in PBS containing 4% PFA for 30 min and then incubated with terminal deoxynucleotidyl transferase (TdT) equilibration buffer (Elabscience) for 30 min at 37 °C. NPCs were incubated in the labeling solution containing TdT enzyme (Elabscience) for 1 h at 37 °C.

[0178] Imaging and cell quantification

[0179] To quantify the number of DCX, SOX2, Ki67, NF, and NeuN-positive cells relative to the total transplanted cells (co-labeled with eGFP and Hoechst), one brain section was selected from every 6 consecutive sections and stereological counting was performed on a Zeiss M1 microscope with stereological analysis system software (MBFBioscience). Briefly, the immunolabeled sections were scanned on a Zeiss M1 microscope, the outline of the transplanted area was manually delineated, and then the corresponding fluorescently labeled cells were counted unbiasedly. The number of cells expressing cleaved-caspase3 relative to the total transplanted cells was calculated using ImageJ software. Each set of data was repeated 4 to 6 times. To quantify the number of cells expressing Brn2, Ctip2, and Foxp2 on the coverslips relative to the total GFP + cells, all coverslips were scanned and recorded with a confocal microscope (Nikon) through a 20× objective lens and then counted using ImageJ software. The data were repeated three times. All data were expressed as mean ± SEM.

[0180] Infarct area analysis and quantification

[0181] The infarct area was determined by GFAP, S100β, CSPG, and Iba1 staining. The fluorescence intensity of the infarct area was measured using ImageJ software and normalized relative to the surrounding intact area. To measure the thickness of the infarct area, 6 sections were randomly selected from 35 brain sections and recorded with a confocal microscope (Nikon). The vertical distance from the central surface to the corpus callosum was measured using ImageJ software. All data were repeated four to six times and expressed as mean ± SEM.

[0182] Behavioral tests

[0183] Mice (n = 9 - 10 per group) were tested for rotarod and grid walking tasks. Behavior was evaluated at -14, 0, 14, and 30 days post-transplantation. For the rotarod test, the fall latency was calculated to assess motor function. For the grid walking test, the deficit was calculated as the number of impaired limbs (right foot) within 10 min.

[0184] Isolation of damaged tissue

[0185] At 2, 14, and 44 days after stroke, animals were anesthetized and perfused with cold PBS. The damaged cortex or graft (area with a radius of 1 mm from the center) was manually dissected under a stereological microscope (Zeiss) and stored at -80 °C.

[0186] Western blot

[0187] NPCs and neurons were washed with PBS and resuspended in RIPA buffer containing protease inhibitors and phosphatase inhibitors. Samples were collected into 1.5 mL tubes and kept on ice for 15 min. For tissue samples, the extracts were sonicated in cold RIPA buffer containing protease / phosphatase inhibitors. Use Quick Start TM All samples were quantified using the Bradford protein assay (Bio-RAD), and then Laemmli buffer (Bio-RAD) was added to each tube. The samples were heated at 95 °C for 5 min and then stored at -80 °C. A total of 15 μg of extract was loaded into each well of a 10% Bis-Tris precast gel for electrophoresis.

[0188] After running at 120 V for 40 min, the proteins were transferred to a PVDF membrane and kept at 400 mA for 30 min. Subsequently, the membrane was blocked in 0.1% TBS-Tween (TBST) containing 5% skim milk for 1 h. Then the membrane was incubated with primary antibodies overnight at 4 °C: 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), anti-GAPDH (1:5000, Thermo Fisher Scientific, MA5-15738). The membrane was washed three times with 0.1% TBST and incubated with the corresponding secondary antibody for 1 h at room temperature. Protein bands were visualized by an enhanced chemiluminescence substrate (Promega) and visualized in a Bio-RAD Chemidoc system. The intensity of all bands was analyzed using ImageJ software and normalized relative to the corresponding GAPDH band.

[0189] Quantitative and statistical analysis

[0190] The data of the stroke group and the transplantation group were normally distributed. Unpaired t-tests were used for comparisons between the two groups. Error bars in all figures represent mean ± SEM. A P-value less than 0.05 was considered statistically significant. All data were analyzed using GraphPad Prism.

[0191] Example 2 - Results

[0192] Combination of fibrinogen and CCR5 antagonist protects transplanted NPCs from apoptosis in the ischemic core

[0193] The cell therapy in this study was for chronic stroke because there is no more spontaneous recovery at this stage. In the chronic stage of stroke, the infarcted area forms lesion cysts or cavities isolated by glial scar tissue and filled with inflammatory cells and their secretions. This environment not only lacks physical and nutritional support for transplanted NPCs but also exerts an inflammatory effect on transplanted NPCs. To change the adverse environment in the infarct core, a mixture / composition was developed, which consists 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 down the release of maraviroc, it was mixed with a hydrogel. Fibrinogen (9 mg / mL) was selected because it remains soluble above 0 degrees Celsius, making it easy to transplant. After injection, fibrinogen is mixed with endogenous thrombin released during surgery and forms a gel. This injectable gel not only stabilizes the transplanted cells but also slows down the dilution of maraviroc, as shown by the release curve of maraviroc ( Figure 1 a and Figure 6 a). The effect of the mixture / composition was evaluated by directly transplanting eGFP-H9-derived cortical NPCs (differentiated from human embryonic stem cells (hESCs), eGFP-H9 hESC line, maintained for 50 days, Figure 6 c-f) into the ischemic core in the absence or presence of maraviroc, fibrinogen, or the mixture / composition two weeks after stroke, and then measuring the viability of the transplanted cells one week later ( Figure 1 b and 1c). Ischemic stroke was induced by photochemical embolism formation in the cerebral cortex of SCID mice, where the ischemic cavity was surrounded by GFAP + and S100β + glial scars at day 14 ( Figure 1 c, Figure 6 b-h), and the cortical collapse occurred at day 30 in the untreated group ( Figure 6i). The ischemic injury induced by photothrombotic occlusion presents a relatively uniform size at similar locations without a penumbra region, providing a consistent model for evaluating the efficacy of cell replacement therapy.

[0194] In the presence of fibrinogen alone or the mixture / composition, transplanted cells identified by GFP were observed in the stroke cavity, while in the groups with maraviroc alone or cells alone, almost no GFP was observed. + Cells or no GFP was observed. + Cells ( Figure 1 c and Figure 7 a). Immunostaining of cleaved-caspase3 showed strong fluorescence in the control, maraviroc, and fibrinogen groups, with diffuse staining in the control and maraviroc groups and discrete staining on individual GFP + cells in the ischemic core of the fibrinogen group ( Figure 1 d and 1e), indicating that the transplanted (GFP + ) cells in the control and maraviroc groups were dead (fragmented), and the individual GFP + cells in the fibrinogen group were dying. In contrast, in the mixture / composition group, almost no GFP + cells were positive for caspase ( Figure 1 d and 1e). Thus, the transplanted NPCs survived in the presence of the mixture / composition.

[0195] Stereological quantification of GFP (overlaying DAPI-labeled nuclei) showed the presence of 1.2×10 5 cells in the mixture / composition group ( Figure 1 f and 1g). More than 26% and 81% of the transplanted cells were SOX2 + and DCX + , indicating that the vast majority of NPCs were in an immature stage and began to differentiate into neurons ( Figure 1 f and 1g). Immunostaining of Ki67 (a marker of cell proliferation) showed that approximately 11% of the GFP + cells in the mixture / composition group were Ki67 + , but almost none in the other three groups ( Figure 7 b and 7c). In summary, the results showed that fibrinogen or maraviroc alone was insufficient to improve the survival of transplanted NPCs. Instead, the combination of fibrinogen and maraviroc supported the survival of NPCs transplanted into the ischemic core.

[0196] Surviving NPCs become mature neurons.

[0197] The environment of the ischemic core normally inhibits the differentiation of transplanted NPCs. To determine whether transplanted NPCs survived longer and what they became, the number and fate of transplanted brains and transplanted NPCs were assessed 30 days after transplantation ( Figure 2 a) Grossly, brains from the sham (stroke without transplantation), control, maraviroc, and fibrinogen groups showed collapsed cortex with little or no GFP in the ischemic area. + cell( Figure 8 a), while the brain from the mixture / combination group showed a smooth surface similar to the contralateral side ( Figure 2 b and 2c). The stroke cavity is demarcated by glial scar tissue; thus, it can be tracked by staining with GFAP and / or S100β. It was observed that GFP-labeled transplanted cells were accurately transplanted into the GFAP-demarcated stromal cavity. + and S100β + In the ischemic core surrounded by glial scar ( Figure 2 b), indicating that the transplanted neurons did not migrate to the peri-infarct area. Strikingly, one month after transplantation, the transplanted GFP + The cells filled the entire stroke cavity ( Figure 2 b, 2c). Serial coronal brain sections further revealed that the transplanted cells filled the infarct area as confirmed by positive staining for the human-specific marker STEM121 ( Figure 2 d), and less than 0.4% of GFP + Cells expressing Ki67 ( Figure 8 b and 8c), which showed that the transplanted NPCs mostly stopped proliferating within one month without overgrowth.

[0198] Immunostaining for mature neuronal markers showed that more than 89% of transplanted cells expressed neurofilament (NF) ( Figure 2 Similarly, 56% of transplanted human (STEM121+) cells were positive for NeuN, another mature neuronal marker ( Figure 2 h, 2i). The ratio of NeuN+ / STEM121+ cells showed a gradient, with more NeuN+ cells in the upper layer of the graft and at the edge of the stroke cavity than in the center ( Figure 2 jm). About 9% of GFP + Cells express SOX9, a marker for astrocytes or their progenitors ( Figure 8 d and 8e). At this stage, GFP + Cells were mostly confined within the boundaries of the stroke cavity ( Figure 2 e), indicating that the transplanted neurons did not migrate to the peri-infarct area. Similarly, 56% of the transplanted (STEM121 +)The cells were positive for another mature neuron marker, NeuN Figure 2 i). NeuN + / STEM121 + The proportion of cells showed a gradient, with more NeuN + cells in the upper layer of the graft and at the edge of the stroke cavity than in the center( Figure 2 j-2m). The initial expression of NF corresponded to the initiation of axons. NeuN is widely used to identify mature neurons with mature axons. Therefore, NF is expressed earlier by neurons than NeuN. NF and NeuN are specifically expressed by neurons rather than glial cells. Although the neurons were located in the ischemic cavity, their neurites (indicated by positive staining for the human marker STEM121 and the glutamatergic neuron marker vGluT1) grew into the undamaged area adjacent to the damaged site( Figure 9 a and b). In addition, STEM121+ neurites co-expressed the presynaptic marker synapsin and the postsynaptic marker PSD95( Figure 9 c), indicating that the transplanted cells developed into mature neurons and formed synapses with host neurons. Collectively, these results suggest that in the presence of the mixture / composition, human NPCs developed into mature neurons within 30 days.

[0199] To evaluate whether cell transplantation contributed to functional improvement, behavioral tests were performed, including the rotarod test and the grid walk test. Although the fall latency in the rotarod test showed improvement in the mixture group( Figure 10 a), the grid walk test did not show a significant difference between the mixture group and the control group( Figure 10 b). Therefore, partial functional recovery was achieved using the mixture 1 month after NPC transplantation.

[0200] Successful transplantation was associated with reduced glial reaction and restored angiogenesis

[0201] In chronic cerebral stroke, glial scar tissue forms around the cavity and the cortex often collapses. In mice receiving transplantation without the mixture / composition, the cortex collapsed 30 days after transplantation and there were few or no GFP + cells present( Figure 3 a). Strong staining for the microglial marker Iba1 and the astrocytic marker GFAP demonstrated a strong glial reaction at and around the ischemic site( Figure 3 a and 3b). Iba1+ microglia / macrophages were present in the lesion site and the penumbra, showing an amoeboid morphology( Figure 3 b and 3d). GFAP + reactive astrocytes accumulated around the infarct to the infarct edge( Figure 3b and 3e), consistent with the robust deposition of chondroitin sulfate proteoglycan (CSPG) around the ischemic area ( Figure 3 c and 3f). In contrast, in mice treated with the mixture / composition, the ischemic area was filled with GFP + cells, and thus the ischemic core did not collapse ( Figure 3 a and 3g). More importantly, the activated microglia and reactive astrocytes aggregated around the ischemic area and its periphery were significantly reduced ( Figure 3 d and 3e). The processes of microglia and astrocytes became thinner ( Figure 3 b). The expression of CSPG showed a significantly reduced level at the boundary of the cavity ( Figure 3 c and 3f), indicating a reduction in glial scar.

[0202] In the peri-infarct area, vascular remodeling contributes to neuronal survival after ischemic stroke. Angiogenesis may also be important for transplanted cells. By immunostaining for laminin (a membrane protein that accumulates in blood vessels), it was found that at 30 days after transplantation, blood vessels penetrated into the graft ( Figure 3 h). Compared with the other four groups in which the vasculature was restricted to the lesion margin ( Figure 8 f), the vascular area in the graft showed a density similar to that in the intact cortex ( Figure 3 i), indicating that the graft reconstructed the ischemic cavity through angiogenesis. These results suggest that the successful survival of the graft was accompanied by reduced inflammatory responses, reduced glial scar, and restored angiogenesis.

[0203] NPC transplantation with the mixture / composition downregulated the expression of CCL and CCR5

[0204] Ischemic injury leads to inflammatory responses, including the production of cytokines and chemokines. Their receptors, including CCR5, are expressed in mature neurons in the peri-infarct area 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 1 (MIP), also known as MIP1-α and MIP1-β, respectively. Maraviroc is an antagonist of the chemokine receptor CCR5 and has been shown to protect mature neurons around the infarct area but not the ischemic core. Western blotting indicated that the expression levels of CCR5 and three ligands were upregulated in the infarct area after stroke ( Figure 4 a and 4b). At day 44 after stroke, the levels of CCR5 and its ligands continued to increase, indicating that in the chronic phase, CCR5 was continuously activated by high concentrations of ligands in the infarct area ( Figure 4 b-4f).

[0205] The next question was whether maraviroc alone, fibrinogen alone, or maraviroc and fibrinogen used together altered the expression of CCR5 or CCL. Western blotting of cortical tissue transplanted on day 44 after ischemic lesion or on day 30 after transplantation ( Figure 4 g) showed that the level of CCR5 was significantly reduced in the mixture / composition group ( Figure 4 h and Figure 4 l). When maraviroc was present alone or fibrinogen was present alone, there were no significant differences in the levels of CCL3, 4, 5, but when the maraviroc and fibrinogen mixture / composition was present, the levels of CCL3, 4, 5 were significantly reduced ( Figure 4 h, 4i, 4j, 4k). The results showed that maraviroc alone or fibrinogen alone could not downregulate the levels of CCR5 or CCL3, 4, 5, but transplantation with the mixture / composition seemed to significantly reduce the presence of CCL, thus blocking the signal transduction between CCR5 in the damaged cortical tissue and CCL produced by inflammatory cells.

[0206] Blocking CCR5 activation feedback reduces NPC apoptosis

[0207] Studies have shown that CCR5 is upregulated in neurons in the penumbra area after stroke, and blocking CCR5 signaling by genetic means or maraviroc (100 mg / kg, intraperitoneal injection daily) can promote the survival of these neurons and their synaptic connections, thus enhancing the behavioral recovery of animals. This raises the question of how maraviroc protects NPCs transplanted into the ischemic core. Immunostaining of CCR5 along neural differentiation showed that CCR5 was highly expressed in the membranes and cytoplasm of SOX2 + NPCs and DCX + immature neurons (day 7), but the fluorescence signal was significantly weakened in mature neurons (day 60) ( Figure 5 a-5c). This was confirmed by Western blotting, showing a gradual decrease in its expression ( Figure 5 d). This result indicates that NPCs and immature neurons are potentially sensitive to inflammatory chemokines present in and around the ischemic cavity.

[0208] Next, NPCs were incubated with three ligands (CCL3, CCL4, and CCL5, 300 ng / mL). Compared with the control group, the proportion of cleaved-caspase3 + cells increased in all three groups ( Figure 11 a and 11b). To simulate the environment in ischemic infarction, a combination of three ligands (100 ng / mL each of the three chemokines) was used to incubate NPCs. A large number of apoptotic and detached neural NPCs were observed after treatment ( Figure 11b and 11c). Interestingly, from day 2 to day 4, incubation of differentiating NPCs with the three ligands induced upregulation of CCR5 in the cells ( Figure 5 e and 5f), and correspondingly increased the proportion of TUNEL + cells ( Figure 5 j and 5k). This result indicates that these chemokines indeed promoted the expression of CCR5 on NPCs and induced apoptosis of NPCs even in the absence of microglia.

[0209] The next step was to investigate whether blocking chemokine signaling could reduce NPC apoptosis. Lentivirus expressing CCR5-shRNA was used to knockdown the expression of CCR5. As shown by immunostaining and western blotting, the expression of CCR5-shRNA, but not that of control shRNA, significantly reduced the expression of CCR5 in NPCs ( Figure 5 g-5i). Correspondingly, in the cultures treated with lentivirus carrying CCR5-shRNA, the number of TUNEL + cells was significantly reduced ( Figure 5 j-5k and Figure 12 a). Similarly, blocking CCR5 with maraviroc also reduced the proportion of TUNEL + NPCs without reducing the expression of CCR5 ( Figure 12 a and 12b). Thus, the expression of CCR5 on transplanted NPCs may be amplified by the inflammatory environment in the ischemic cavity, and blocking the CCR5 pathway by maraviroc or RNA interference (RNAi) can protect susceptible NPCs from apoptosis ( Figure 5 l).

[0210] Stroke mice transplanted with NPCs recovered from motor deficits

[0211] At 14 days after stroke, human ESC-derived neural progenitor cells were transplanted into the damaged site. In rodents, 14 days after stroke corresponds to the chronic phase in humans. This study showed that the transplanted cells survived and differentiated into neurons one month after transplantation. As time elapsed after transplantation, the presence of human axons was observed in the brainstem and spinal cord. Correspondingly, the stroke animals recovered from motor deficits. By 12 months after transplantation, although the transplanted human neurons remained in the stroke area, their axons (labeled by human-specific neural cell adhesion molecule (hNCAM)) extended into the striatum ( Figure 13 D), passed through the internal capsule ( Figure 13 E) and extended along the pyramidal tract ( Figure 13 B). When reaching the medulla oblongata, the human axons crossed to the contralateral side of the pyramidal tract ( Figure 13C). In the spinal cord, human axons continue to extend downward along the contralateral pyramidal tract, as low as the thoracic level. This is evident in the positively immunostained human nerves in the pyramidal tract and adjacent region (T1). Figure 14 ) These results suggest that transplanted human cortical neurons not only survive and mature at the stroke site, but also grow along the corticospinal tract all the way to the spinal cord, thus reconstructing the corticospinal tract damaged by the stroke. This explains why stroke animals recover from motor deficits.

[0212] Example 3 - Discussion

[0213] A mixture / composition mainly composed of maraviroc and fibrinogen was developed to support the survival of transplanted NPCs in the ischemic core. In the presence of the mixture / composition, human NPCs transplanted into the ischemic cyst survived and then divided and matured, reconstructing the damaged cortex in the stroke model at day 30. This was achieved by blocking the signal transduction between inflammatory chemokines in the ischemic lesion and the high levels of CCR5 on NPCs. The transplanted NPCs survived and matured in the ischemic core, accompanied by a significant reduction in glial scarring and angiogenesis of the graft. Specifically, when assayed at 7 days post-transplantation, NPCs in the mixture / composition group survived. Over the next 3 weeks, the surviving NPCs proliferated and filled the stroke cavity. Importantly, most transplanted NPCs differentiated into NeuN-positive neurons one month after transplantation. In the group using only fibrinogen, there were some surviving cells that did not proliferate and became GFAP-positive astrocytes. These results suggest that the mixture / composition supports the survival of transplanted NPCs in other adverse environments. In addition, host endothelial cells penetrated into the transplanted tissue and formed blood vessels in the graft, supporting the long-term survival and maturation of transplanted neurons. In addition, glial scarring, indicated by the increased expression of GFAP at the cavity border, was significantly reduced. Six months after transplantation, the transplanted neurons projected their axons to the brainstem and spinal cord and to the contralateral cortex, reconnecting the ischemic cortex to the rest of the brain.

[0214] This appears to be achieved by the mixture / composition disclosed herein, which supports the survival of NPCs in the inflammatory lesion cavity and promotes the neuronal differentiation of surviving NPCs. Fibrinogen can form a gel and serve as a scaffold to stabilize the transplanted cells at the injury site. It also supports the growth of transplanted NPCs. Maraviroc, an antagonist of C-C chemokine receptor type 5 (CCR5), blocks the signal transduction between inflammatory chemokines in the ischemic lesion and the high levels of CCR5 on NPCs, thus blocking the activation of CCR5 expressed by NPCs, reducing apoptosis and promoting the maturation of transplanted NPCs. The transplanted NPCs survived and matured in the ischemic core, accompanied by a significant reduction in glial scarring and angiogenesis of the graft.

[0215] After ischemic stroke, inflammatory cell infiltration occurs and inflammatory cytokines, such as CCL, are released. Reactive glial cells form a wall around the lesion site to prevent the spillage of pro-inflammatory mediators. Therefore, as observed, the inflammatory environment in the ischemic cavity persists. As a result, few of the NPCs transplanted into the cavity survive, and the surviving NPCs (if any) tend to differentiate into astrocytes. It is unclear which inflammatory pathway induces the death of transplanted NPCs in the ischemic core. In the penumbra, CCR5 inhibits the expression of PKA and CREB on neurons, leading to dendritic spine loss and increased neuronal death. However, this does not explain the low survival rate of transplanted NPCs in the ischemic core. In the current study, it was found that NPCs express high levels of CCR5, highlighting the sensitivity of NPCs to the inflammatory environment. Its ligands CCL3 / 4 / 5, secreted by blood-derived cells infiltrating during stroke, activate microglia and astrocytes. Reactive astrocytes and activated microglia also produce CCL3 / 4 / 5 as well as other cytokines, forming a cascade of inflammatory responses. Together, they induce apoptosis of transplanted NPCs that express their receptor CCR5. Worse still, the CCL present in the inflammatory environment further stimulates the expression of CCR5. This explains why few of the NPCs transplanted into the ischemic cavity survive. In fact, no survival of NPCs transplanted into the ischemic core was observed. Therefore, most existing conventional experimental cell transplantation therapies target healthy brain regions adjacent to ischemic lesions to avoid the toxic environment in the ischemic lesions. However, transplantation into the penumbra causes additional damage and poses a substantial risk to patients. Therefore, a method is needed to protect NPCs transplanted into inflammatory ischemic lesions. The mixture not only directly blocks the CCL-CCR5 pathway on NPCs but also blocks the CCL-CCR5 pathway on reactive glial cells, thereby directly and indirectly protecting transplanted NPCs by reducing the production of CCL.

[0216] The protection of transplanted NPCs mainly targets neurotrophic support, such as overexpressing SUMO in NPCs, hypoxic treatment, co-transplantation, and using biomaterials crosslinked with growth factors. However, due to the limited number of surviving cells, these methods cannot fill and reconstruct the damaged brain. Since inflammation is the main cause of cell death, blocking inflammatory signal transduction is sufficient to prevent cell death. In fact, it was found that blocking the CCL-CCR5 signal transduction in NPCs by genetic means (RNAi) or a chemical antagonist of CCR5, namely maraviroc, is sufficient to prevent the apoptosis of NPCs even in an in vitro system. A recent study showed that administering maraviroc (100 mg / kg, intraperitoneal injection daily) saved neurons in the perifocal area from death. Due to the lack of blood flow in the ischemic cavity, peripheral administration is unlikely to affect NPCs transplanted into the lesioned cyst or cavity. Therefore, in this study, NPCs were transplanted in the presence of maraviroc, but this was not sufficient to save any of the transplanted cells. This may be due to the rapid dilution and / or degradation of maraviroc (half-life: 14-18 h). In fact, when maraviroc is combined with fibrinogen, fibrinogen forms a degradable gel in the presence of thrombin or Ca ++ and slows down the release of maraviroc, and the transplanted NPCs survive, even though fibrinogen itself does not show support for the survival of transplanted NPCs. Fibrinogen is neurotrophic and gelifiable. The fact that fibrinogen alone does not support the survival of NPCs indicates that simply retaining transplanted cells in the lesioned cyst or cavity by neurotrophic fibrinogen may not be sufficient to support cell survival. Mitigating inflammatory damage in this environment may be necessary.

[0217] Surviving human NPCs appear to proliferate and fill the entire lesioned cyst or cavity within 30 days after transplantation. Notably, the glial scar around the ischemic cavity is greatly reduced, as evidenced by a significant decrease in GFAP and IBA1 immunoreactivity, and angiogenesis of the graft indicated by vessels labeled with laminin occurs. In addition to the effect of maraviroc, this tissue alteration may be the result of complex interactions between transplanted cells and host cells. Perhaps most strikingly, the vast majority of transplanted NPCs become post-mitotic neurons within 30 days, as indicated by their NeuN and NF expression. Human cortical NPCs tend to proliferate for a long time before differentiating into mature neurons, which explains the proliferation of surviving NPCs and their filling of the lesioned cyst or cavity within 30 days. The rapid differentiation / maturation may be due to the lack of growth factors in the environment. This is because past studies have shown that spinal cord NPCs transplanted into the lesioned cyst or cavity of the injured spinal cord remain in the precursor state for months in the presence of high concentrations of growth factors (thousands of times their physiological concentration), resulting in overgrowth. Thus, the mixtures / compositions disclosed herein provide a basal medium for safe cell transplantation therapy, facilitating the survival and differentiation of transplanted cells, i.e., NPCs. Depending on the number and developmental stage of donor cells and the size of the lesion to be repaired, growth factors such as FGF2 can be supplemented to promote proliferation, or neurotrophic factors such as BDNF and GDNF to promote further survival and maturation, or notch inhibitors such as compound E to enhance cell cycle exit. While the basal mixtures / compositions can be modified to suit the needs according to the nature of the disease and the nature of the NPCs, the mixtures / compositions disclosed herein provide the possibility of repairing gap lesions such as stroke and other inflammatory neuropathological conditions by cell transplantation therapy.

[0218] Industrial applicability

[0219] It will be apparent to those skilled in the art that various other modifications and adaptations of the present invention are obvious after reading the foregoing disclosure, and all such modifications and adaptations are within the scope of the appended claims without departing from the essence and scope of the present invention.

Claims

1. 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 C-C chemokine receptor type 5 (CCR5) antagonist.

2. The composition according to claim 1, wherein the gel-forming molecule is selected from the group consisting of fibrinogen, agarose, collagen, gelatin, chitosan, alginate, fibrin, hyaluronic acid, laminin, and degradable polymers, and the degradable polymers are 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 according to claim 2, wherein the gel-forming molecule is fibrinogen.

4. The composition according to any one of claims 1 to 3, wherein the CCR5 antagonist is a small molecule selected from the group consisting of maraviroc, phloretin, TAK-220, nelfinavir, 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 according to claim 4, wherein the CCR5 antagonist is maraviroc.

6. The composition according to 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. The composition according to any one of claims 1 to 6, wherein the site of nerve injury or disease is a site resulting from a nerve injury or disease characterized by inflammation, and the nerve injury or disease 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).

8. The composition according to any one of claims 1 to 7, wherein the composition further comprises CaCl2.

9. The composition according to any one of claims 1 to 8, wherein the composition further comprises thrombin.

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

11. The composition according to claim 3, wherein the concentration of fibrinogen is 5 mg / mL - 30 mg / mL.

12. The composition according to claim 5, wherein the concentration of maraviroc is 3 mg / mL - 50 mg / mL.

13. A method of treating a nerve injury or disease in a subject, comprising: (a) mixing NPCs with the composition according to any one of claims 1 to 12; and (b) Administer the mixture of the NPC and the composition to the site of nerve injury or disease of the subject, thereby supporting the survival and differentiation of the NPC.

14. The method according to claim 13, wherein the NPC is 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.

15. The method according to claim 13 or 14, wherein the nerve 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 according to any one of claims 13 to 15, wherein the number of NPCs to be mixed with the composition is 50,000 - 500,000.

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

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

19. Use of a mixture of an NPC and the composition according to any one of claims 1 to 12 in the preparation of a medicament for treating nerve injury or disease of a subject, wherein the mixture is administered to the site of nerve injury or disease of the subject, thereby supporting the survival and differentiation of the NPC.

20. The use according to claim 19, wherein the NPC is 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.

21. The use according to claim 19 or 20, wherein the nerve 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 to be mixed with the composition is 50,000 - 500,000.

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

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

25. A kit for supporting the survival and differentiation of NPCs transplanted into the site of nerve injury or disease, the kit comprising: (a) The composition according to any one of claims 1 to 12; (b) Artificial cerebrospinal fluid (a-CSF); (c) CaCl2; and (d) Thrombin.

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