Reshaping the hematopoietic niche to reconstitute immunity

Abstract

The present invention relates to remodeling the hematopoietic niche to reconstitute immunity. In particular, the present invention discloses compositions and related methods for remodeling the bone marrow stroma using a scaffold material (e.g., a porous alginate hydrogel scaffold) containing one or more cell differentiation factors and one or more growth factors. These methods and compositions promote the formation of ectopic nodules or sites that can improve engraftment of transplanted cells and selectively drive the development of lymphocytes and reconstitution of adaptive immunity following hematopoietic stem cell transplantation.

Description

[0001] This application is a divisional application of the application filed on February 6, 2017, with international application number PCT / US2017 / 016729, which entered the Chinese national phase on August 3, 2018, with application number 201780009851.4 and invention title "Reshaping Blood Nests to Rebuild Immunity".

[0002] Related applications

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 292,288, filed February 6, 2016, the entire teachings of which are incorporated herein by reference.

[0004] Government funding

[0005] This invention was developed with government support, under license numbers NIH HL129903, NIH EB015498, and NIH EB014703 issued by the National Institutes of Health and NSF 1000099416 issued by the National Science Foundation. The government owns certain rights to this invention. Background Technology

[0006] Long-term immunodeficiency in patients receiving hematopoietic stem cell transplantation (HSCT) remains one of the most serious obstacles to controlling life-threatening blood or bone marrow diseases such as multiple myeloma and leukemia. Prior to transplantation, recipients undergo conditioning cytotoxic radiation and chemotherapy regimens to destroy diseased cells. A side effect of these conditioning methods is severe lymphopenia due to the destruction of T and B cells in the adaptive immune system. Severe post-transplant immunodeficiency, characterized by a sharp decrease in the number and diversity of T and B cells, can last for one to two years. Immunodeficiency-related severe opportunistic infections (approximately 30%), cancer relapse (acute myeloid leukemia > 50%), and graft-versus-host disease (GVHD) (approximately 40%) are the most common complications and causes of morbidity and death in patients receiving HSCT.

[0007] There is a need for novel compositions and methods that can be used to improve immune system remodeling after HSCT. There is also a need for compositions and methods that can reduce the risks associated with HSCT and improve patient outcomes. Summary of the Invention

[0008] This article discloses novel compositions and related methods that can be used, for example, to help rebuild the immune system of a subject after stem cell transplantation. These compositions can be administered to the subject to increase the engraftment of transplanted stem cells and progenitor cells, thereby helping to rebuild the subject's immune system.

[0009] In some embodiments, the compositions disclosed herein comprise one or more scaffold materials (e.g., porous implantable scaffold materials) that can be applied to or otherwise implanted in a subject (e.g., subcutaneously implanted in or around one or more sites in the subject's lymph nodes). These compositions may also comprise one or more growth factors, one or more homing factors, and one or more differentiation factors.

[0010] In some aspects, the scaffold material is or comprises a hydrogel (e.g., a cryogel). In some embodiments, the scaffold material is osteoinductive. In some embodiments, the scaffold material comprises alginate (e.g., anionic alginate). In some embodiments, the scaffold material is selected from: polylactic acid, polyglycolic acid, PLGA polymers, alginate and alginate derivatives, polycaprolactone, calcium phosphate-based materials, gelatin, collagen, fibroin, hyaluronic acid, laminin-rich gels, agarose, natural and synthetic polysaccharides, polyamino acids, peptides, polyesters, polyanhydrides, polyphosphazine, poly(vinyl alcohol), poly(epoxyalkylene), poly(allylamine) (PAM), poly(acrylate), modified styrene polymers, pluronic polyol, polyoxamer, poly(uronic acid), poly(vinylpyrrolidone), and any combination or copolymer thereof.

[0011] The compositions and scaffold materials disclosed herein can be used as carriers or mediators for delivering one or more growth factors (e.g., delivering one or more growth factors in vivo). Upon application or implantation of the composition, the one or more growth factors contained in the composition promote the formation of tissue (e.g., bone tissue) on or around the applied scaffold material, thereby forming nodules. In some aspects, the one or more growth factors comprise bone morphogenetic proteins (BMPs) (e.g., BMP-2). For example, one or more growth factors may be selected from: BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-14, TGF-β, IGF-1, FGF-2, and PDGF. In some embodiments, one or more growth factors may be encapsulated in the scaffold material (e.g., encapsulated and released from the scaffold material within approximately 7-14 days). In some embodiments, such one or more growth factors (e.g., BMP-2) are released from the scaffold material over an extended period of time (e.g., approximately 7-30 days or longer, approximately 17-18 days).

[0012] The compositions and scaffold materials disclosed herein can also be used as carriers or mediators of one or more differentiation factors. In some aspects, the differentiation factors of consideration induce or promote the differentiation of stem cells or progenitor cells (e.g., transplanted HSCs) into one or more desired cell types. For example, one or more differentiation factors may be incorporated into the compositions disclosed herein to promote the differentiation of transplanted stem cells or progenitor cells into lymphoid lineage cells. In some embodiments, one or more differentiation factors bind to Notch receptors (e.g., one or more differentiation factors may bind to Notch receptors selected from Notch-1, Notch-2, Notch-3, and Notch-4). In some aspects, one or more differentiation factors are selected from δ-sample 1, δ-sample 3, δ-sample 4, Jagged1, and Jagged2.

[0013] In some embodiments, one or more differentiation factors comprise cytokines (e.g., cytokines selected from interleukin-7 (IL-7) and interleukin-15 (IL-15)). In some embodiments, one or more cytokines may be encapsulated in the scaffold material. In some aspects, one or more cytokines are released from the scaffold material over an extended period of time (e.g., about 7-30 days or longer, about 17-18 days).

[0014] In some embodiments, one or more differentiation factors are covalently bound to the scaffold material. For example, the differentiation factor may be covalently bound to the alginate backbone and retained in the nodules formed after implantation of the composition in the subject, rather than being released from the alginate scaffold material. By covalently binding or coupling the differentiation factor to the scaffold material, this differentiation factor will be retained within the nodules formed after administration of the composition to the subject, and thus can be used to promote the differentiation of transplanted stem cells or progenitor cells, as contemplated herein. In some embodiments, one or more differentiation factors are bound to the scaffold material using a chemical method involving N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Any method of covalently binding or coupling differentiation factors known in the art can be used, and is not limited thereto. See “Bioconjugate Techniques (Third Addition)”, Greg T. Hermanson, Academic, Greg T. Hermanson, Academic Press, 2013.

[0015] In some embodiments, the compositions and scaffold materials disclosed herein comprise one or more homing factors. In some aspects, including these homing factors in the compositions disclosed herein promotes the homing of transplanted stem cells and / or progenitor cells (e.g., HSCs) to the implanted composition or nodule. In some aspects, these homing factors promote the infiltration of transplanted stem cells or progenitor cells (e.g., HSCs) into the implanted composition or nodule. In some embodiments, one or more homing factors comprise stem cell differentiation factor (SDF-1). In some embodiments, one or more homing factors are encapsulated in the material. In some embodiments, one or more homing factors are released from the material over an extended period of time (e.g., about 7-30 days or longer, about 17-18 days).

[0016] In some embodiments, one or more growth factors, one or more homing factors, and one or more differentiation factors are combined with the scaffold material in amounts of about 0.01 nmol to 1000 nmol, about 0.1 nmol to 100 nmol, or 1 nmol to about 10 nmol.

[0017] In some embodiments, the present invention relates to methods for assisting or supporting the reconstruction of the immune system in a subject in need, methods comprising the step of administering to the subject a composition comprising a scaffold material, said composition comprising: one or more growth factors that promote tissue formation on or around the administered scaffold material to form nodules; one or more homing factors that promote the infiltration of transplanted stem cells or progenitor cells into the nodules; and one or more differentiation factors that promote the differentiation of transplanted stem cells or progenitor cells into lymphoid lineage cells, thereby assisting or supporting the reconstruction of the subject's immune system. In some embodiments, the subject has undergone cytotoxic radiation and / or chemotherapy to treat a blood disorder.

[0018] In some embodiments, the invention disclosed herein relates to methods for forming ectopic hematopoietic stem cell niches in subjects in need, methods comprising the step of administering to the subject a composition comprising: one or more growth factors that promote tissue formation on or around the administered scaffold material to form a nodule; one or more homing factors that promote the infiltration of transplanted hematopoietic stem cells into the nodule; and one or more differentiation factors that promote the differentiation of transplanted stem cells or progenitor cells into one or more lymphatic or bone marrow lineage cells, thereby forming ectopic hematopoietic stem cell niches in the subject.

[0019] In other embodiments, the present invention relates to a method for improving the implantation of transplanted hematopoietic stem cells into stem cell nests of a subject in need, the method comprising administering to the subject a composition comprising a scaffold material, the composition comprising: one or more growth factors that promote tissue formation on or around the applied scaffold material to form nodules; one or more homing factors that promote the infiltration of transplanted hematopoietic stem cells into nodules; and one or more differentiation factors that promote the differentiation of transplanted stem cells or progenitor cells into one or more lymphoid or bone marrow lineage cells (e.g., one or more of CD4+, CD8+, and Mac-1+ / GR-1+ lymphoid or bone marrow lineage cells), thereby improving the implantation of transplanted hematopoietic stem cells into stem cell nests of the subject.

[0020] In some embodiments, the invention disclosed herein relates to methods for increasing the number or volume of transplanted stem cell and progenitor cell implantation sites in a subject in need, comprising administering to the subject a composition comprising a scaffold material, the composition comprising: one or more growth factors that promote tissue formation on or around the applied scaffold material to form nodules; one or more homing factors that promote the infiltration of transplanted stem cells and progenitor cells into nodules; and one or more differentiation factors that promote the differentiation of transplanted stem cells or progenitor cells into one or more lymphoid or bone marrow lineage cells (e.g., one or more of CD4+, CD8+, and Mac-1+ / GR-1+ lymphoid or bone marrow lineage cells), thereby increasing the number of transplanted stem cell and progenitor cell implantation sites in the subject.

[0021] In some aspects, the scaffold material considered for use in the method according to the invention comprises a hydrogel material (e.g., a cryogel material). In some embodiments, the scaffold material comprises alginate (e.g., a scaffold material comprising anionic alginate). In some embodiments, the scaffold material is osteoinductive. In some embodiments, the scaffold material is selected from: polylactic acid, polyglycolic acid, PLGA polymers, alginate and alginate derivatives, polycaprolactone, calcium phosphate-based materials, gelatin, collagen, fibroin, hyaluronic acid, laminin-rich gels, agarose, natural and synthetic polysaccharides, polyamino acids, peptides, polyesters, polyanhydrides, polyphosphazene, poly(vinyl alcohol), poly(epoxyalkylene), poly(allylamine) (PAM), poly(acrylate), modified styrene polymers, pululanic polyols, poloxamer, poly(uronic acid), poly(vinylpyrrolidone), and any combination or copolymer thereof.

[0022] The compositions and methods disclosed herein generally encompass the step of administering the composition to a subject. In some aspects, such an administration step includes implanting the composition into the subject (e.g., the composition may be implanted subcutaneously). For example, in some embodiments, the compositions disclosed herein may be administered subcutaneously to a subject at or around a lymph node before stem cell transplantation (e.g., approximately 5, 7, 10, 14, 18, 21, 24, 28, 30, 35, 42 days or longer prior to stem cell transplantation).

[0023] The composition used according to any of the foregoing methods may contain one or more growth factors. For example, these growth factors (e.g., BMP-2) may be encapsulated in a scaffold material and promote the formation of nodules (e.g., bone nodules) in or around the applied composition. In some aspects, one or more growth factors comprise bone morphogenetic proteins (BMPs). In some embodiments, one or more growth factors are selected from BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, and BMP-14. In some aspects, one or more growth factors are encapsulated in a material. In some aspects, one or more growth factors are released from the material over an extended period of time (e.g., about 7-30 days or longer).

[0024] The composition used according to any of the foregoing methods may also contain one or more differentiation factors. These differentiation factors can be used to promote the differentiation of transplanted stem cells and progenitor cells (e.g., transplanted HSCs) in vivo. For example, in some aspects, one or more differentiation factors promote lymphopoiesis of transplanted stem cells and progenitor cells (e.g., transplanted HSCs) in vivo.

[0025] In some aspects, one or more differentiation factors comprise a composition that binds to a Notch receptor. In some aspects, the Notch receptor is selected from Notch-1, Notch-2, Notch-3, and Notch-4. In some embodiments, one or more differentiation factors are selected from δ-sample 1, δ-sample 3, δ-sample 4, Jagged1, and Jagged2.

[0026] In some implementations, one or more differentiation factors comprise cytokines. For example, one or more differentiation factors may be selected from the group of cytokines consisting of IL-7 and IL-15.

[0027] In some embodiments, one or more differentiation factors (e.g., cytokines) are encapsulated in the material. In some embodiments, one or more differentiation factors (e.g., cytokines) are released from the material over approximately 7-30 days. Alternatively, in some embodiments, one or more differentiation factors are covalently bound to the material.

[0028] The composition used according to any of the foregoing methods may also contain one or more homing factors. In some embodiments, the one or more homing factors comprise stem cell differentiation factor (SDF-1). In some embodiments, one or more homing factors are encapsulated in the material. In some embodiments, one or more homing factors are released from the material over approximately 7-30 days.

[0029] The compositions and methods disclosed herein are suitable for administration or implantation in subjects (e.g., subjects in need). For example, these compositions and methods can be used to treat subjects who have undergone stem cell transplantation and / or immunocompromised subjects.

[0030] The above and many other features and accompanying advantages of the present invention will become better understood with reference to the following detailed description of the invention. Attached Figure Description

[0031] The patent or application document contains at least one color drawing. A copy of the patent or application disclosure with color drawing will be provided by the competent authority upon request and payment of the necessary fees.

[0032] Figure 1 The general approach of one embodiment of the present invention is described. For example... Figure 1 As described, an injectable protein-polymer hydrogel that forms bone nodules recruits transplanted hematopoietic stem cells (HSCs) in vivo. The recruited HSCs acquire differentiation factors present in the hydrogel, driving their differentiation into lymphocytes.

[0033] Figures 2A-2E Depicting ectopic bone nodules formed according to the present invention. Figure 2A This demonstrates the release of growth factor (BMP-2) encapsulated within the hydrogel, and Figure 2B (Above) The release of growth factor (BMP-2) encapsulated in a hydrogel and released within approximately one to two weeks is illustrated in a diagram. Figure 2B (The image shows the release of both BMP-2 and differentiation factor δ-like 4 (DLL-4) from the hydrogel within approximately one to two weeks.) Figure 2B (Below) shows the cumulative amount of BMP-2 and differentiation factor interleukin-7 (IL-7) released from the hydrogel over approximately one to two weeks. Figure 2C The subcutaneous bony tubercles are depicted (red arrows). Figure 2D Three-dimensional micro-computed tomographic images of the bony tubercle were depicted. Figure 2E Histological sections of the excised bone tubercle stained with Safranin-O depicting bone alginate and bone marrow.

[0034] Figure 3A and 3B Depicting lymphocyte proliferation mediated by the hydrogel according to the present invention. Figure 3A The study demonstrated that isolated LKS cells cultured in vitro exhibited differentiation (n=9) in the presence of a hydrogel forming at the bone tubercle and in the presence of fixed tethered DLL-4. P<0.001, P < 0.01, ns = not significant. Figure 3B This indicates that, in vivo, ectopic bone nodules accelerate the regeneration of T cells (CD3+) and B cells (B220+) in sublethal irradiated mice. The regeneration kinetics of the bone marrow cell (Mac-1+Gr-1+) population remained unchanged.

[0035] Figure 4A and 4B Describe the characterization of immune reconstitution after hematopoietic stem cell transplantation (HSCT). For example... Figure 4A As shown, in the case of grafts with T-cell and B-cell anemia, hydrogels containing BMP-2 / DLL-4 significantly accelerated the recovery of T-cells and B-cells after HSCT. Figure 4B The analysis results of the immune repertoire 2 weeks after HSCT were depicted, and indicated that a large VJ recombination event occurred in CD3+ T cells with BMP-2 / DLL-4 hydrogel.

[0036] Figure 5 This displays standard HBSC treatment for hematologic disorders, including leukemia and myeloma. The top row shows patients with hematologic disorders receiving radiation therapy to eradicate host blood cells, followed by the reconstruction of the blood and immune system with hematopoietic stem cell grafts. The bottom row shows blood samples from patients with hematologic disorders; normal and irradiated bone marrow; and the re-proliferation of bone marrow cells in weeks after hematopoietic stem cell transplantation and the re-proliferation of T cells and B cells months to years after transplantation.

[0037] Figure 6 A schematic diagram is shown in which HSCs and the hydrogel with DLL-4 described herein are implanted into mice to accelerate immune reconstitution.

[0038] Figure 7 The composition of the hydrogel as described herein is shown. The top row shows DLL4-PEG. 2k -MA and BMP-2. The middle row illustrates the methylation of alginate and PEG using the EDC / NHS chemical method and 2-aminoethyl methacrylate (AEMA). The bottom row illustrates the formation of macroporous cryogels as described herein via cryo-polymerization.

[0039] Figure 8A shows a schematic diagram of in vitro culture and analysis of hematopoietic stem cells. Figure 8B illustrates the degree of functionalization with COOH and the relative cell abundance of T cells, B cells, bone marrow cells, CLPs, and CMPs.

[0040] Figure 9 The different CLP / T cell kinetics in vitro are shown to be DLL-4 dependent. The left panel provides data on cells in contact with a hydrogel containing BMP-2 and DLL-4. The right panel provides data on cells in contact with a hydrogel containing BMP-2.

[0041] Figure 10 This diagram illustrates cell transplantation in irradiated mice using GFP+ cells.

[0042] Figure 11 The results showed that BMP-2 release increased the number of transplanted cells in the cryogel.

[0043] Figure 12 The results showed that BMP-2 and DLL-4 increased the number of CLP cells in the cryogel.

[0044] Figure 13A shows BMP-2-induced ectopic bone marrow nodules. Figure 13B is an image of ectopic bone marrow nodules grown using the methods disclosed herein. Figure 13C is a photograph of bone nodules in subcutaneous tissue grown using the methods disclosed herein.

[0045] Figure 14 The following mice are shown to have the number of T cells, B cells, and bone marrow cells 0-60 days after irradiation: mice with only a graft (left), mice with a graft plus a hydrogel containing BMP-2 (middle), and mice with a graft plus a hydrogel containing BMP-2 and DLL-4 (right).

[0046] Figure 15 This indicates that sequencing of CDRs 1-3 in TCRβ can provide a snapshot of cellular diversity.

[0047] Figure 16 TCR library analysis is shown in the cases of no irradiation, graft-only, graft-with hydrogel containing BMP-2, and graft-with hydrogel containing both BMP-2 and DLL-4.

[0048] Figure 17A shows a schematic diagram of sjTREC analysis used to measure thymic output. Figure 17B The sjTREC per milligram of thymus is shown in the cases of no irradiation, graft alone, graft plus hydrogel with BMP-2, and graft plus hydrogel with BMP-2 and DLL-4.

[0049] Figure 18A shows a schematic diagram of a sublethal viral injection attack one month after immune reconstitution. Figure 18BThis shows the survival rates of mice after viral challenge in mice that were not irradiated, used only with grafts, used with grafts plus a hydrogel with BMP-2, and used with grafts plus a hydrogel with BMP-2 and DLL-4. Detailed Implementation

[0050] A fundamental challenge following hematopoietic stem cell transplantation (HSCT) involves generating a novel immune response while avoiding an overactive response that could lead to autoimmune disease. At the stem cell level, the immune system originates from hematopoietic stem cells (HSCs), which reside in the bone marrow nests and comprise cells and the surrounding matrix. In addition to its role as a major lymphoid organ by supporting lymphoid development, the bone marrow acts as a host for various mature lymphocyte types. The bone marrow influences the regenerative capacity of HSCs and their ability to differentiate into immune cells, providing a progenitor cell population for new T cells and B cells.

[0051] The conditioning process in hematopoietic stem cell transplantation (HSCT) impairs the bone marrow nest and its ability to support donor cell engraftment, thereby directly affecting the reconstruction of the immune system. Therefore, immunomodulatory small molecules, therapeutic proteins, and infusion of purified donor T cells are sometimes used to temporarily increase the absolute number of immune cells in the transplant recipient; however, these methods rely on clonal expansion and donor cell activation and cannot restore the diversity of the T-cell and B-cell repertoire.

[0052] Compared to endogenous bone marrow, the present invention generally relates to compositions and methods involved in the formation of ectopic bone marrow nests or nodules, which are formed after the compositions disclosed herein are administered or implanted into a subject (e.g., a mammalian subject in need). For example, in some aspects, the present invention relates to compositions comprising a porous implantable scaffold material and one or more differentiation factors that embodied lymphocyte proliferation and promoted the differentiation of transplanted HSCs into lymphoid lineage cells (CLPs), thereby helping to rebuild the subject's immune system. Thus, the endogenous generation of naïve immune cells capable of producing an immune response against appropriate antigens provides broad and long-term immunity.

[0053] The compositions disclosed herein comprise one or more scaffold materials (e.g., porous implantable scaffold materials) that can be applied to or otherwise implanted into a subject. For example, in some embodiments, the compositions disclosed herein comprise one or more growth factors that can be loaded into or encapsulated by the scaffold material, and after application of such a composition to a subject (e.g., an immunocompromised subject), the composition promotes the formation of tissue (e.g., bone tissue) on or around the applied scaffold material to form nodules as ectopic bone marrow nests. Thus, in some aspects, the scaffold materials disclosed herein provide a delivery medium for one or more growth factors, differentiation factors, homing factors, cytokines, chemokines, and any other agents.

[0054] In some embodiments, the scaffold material comprises a polymer (e.g., a three-dimensional polymer system). In some embodiments, the scaffold material is osteoinductive. In some aspects, the scaffold material is or comprises alginate (e.g., anionic alginate). In some embodiments, the scaffold material is in hydrogel form.

[0055] In some embodiments, the scaffold material is in the form of a cryogel. Cryogels are a class of materials with a highly porous, interconnected structure, produced using cryotropic gelation / cryogelation techniques. Cryogelation is a technique that involves a polymerization-crosslinking reaction in a quasi-freezing reaction solution. During the freezing of the macromolecular (e.g., MA-alginate) solution, macromonomers and initiator systems (e.g., APS / TEMED) are expelled from the ice concentrate within the channels between ice crystals, allowing the reaction to occur only in these unfrozen liquid channels. After polymerization and after the ice melts, a porous material is produced whose microstructure is a negative replica of the formed ice. The ice crystals act as porogens. The pore size is adjusted by varying the temperature of the cryogelation process. For example, cryogelation is typically performed by rapidly freezing the solution at -20°C. Lowering the temperature to, for example, -80°C will produce more ice crystals and smaller pores. In some embodiments, the cryogel is prepared by cold polymerization of at least methacrylated (MA)-alginate and MA-PEG. In some embodiments, the cryogel is prepared by cold polymerization of at least MA-alginate, a differentiation factor, and MA-PEG. In some embodiments, the differentiation factor further comprises a chain (e.g., PEG, PEG-PEG-PEG). 2k ) and MA groups. In some embodiments, the differentiation factor is DLL4-PEG. 2k-MA. In some embodiments, cold polymerization is carried out in the presence of a reagent to be encapsulated in a cryogel. In some embodiments, the reagent is one or more growth factors, differentiation factors, homing factors, cytokines, and chemokines. In some embodiments, the reagent is BMP (e.g., BMP-2).

[0056] Cryogels may contain at least 75% pores, such as 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more pores. These pores are interconnected. This interconnectivity allows water (and other compositions, such as cells and compounds) to enter and exit the structure. In a fully hydrated state, the composition contains at least 90% water (e.g., 90-99%, at least 92%, 95%, 97%, 99%, or more). For example, at least 90% (e.g., at least 92%, 95%, 97%, 99%, or more) of the cryogel volume is composed of liquid (e.g., water) contained in the pores. In compressed or dehydrated hydrogels, up to 50%, 60%, or 70% of the water is absent; for example, cryogels contain less than 25% (20%, 15%, 10%, 5%, or less) of water.

[0057] The cryogel of the present invention can contain pores large enough to allow cells to pass through. For example, the cryogel contains pores with diameters of 20-500 μm (e.g., 20-300 μm, 30-150 μm, 50-500 μm, 50-450 μm, 100-400 μm, 200-500 μm). In some cases, the hydration pore size is 1-500 μm (e.g., 10-400 μm, 20-300 μm, 50-250 μm).

[0058] In some embodiments, the cryogel is further functionalized by adding functional groups selected from the following: amino, vinyl, aldehyde, thiol, silane, carboxyl, azide, alkyne. Alternatively, the cryogel is further functionalized by adding another crosslinking agent (e.g., multi-arm polymer, salt, aldehyde, etc.). The solvent can be aqueous and, in particular, acidic or basic. Aqueous solvents may include solvents miscible with water (e.g., methanol, ethanol, DMF, DMSO, acetone, dioxane, etc.). In some embodiments, one or more functional groups are added to the components of the cryogel (e.g., alginate, PEG) prior to cryogeling. Cryocrosslinking is performed in a mold, and the injectable cryogel can be biodegradable. Pore size can be controlled by selecting the main solvent used, incorporating pore-forming agents, applying freezing temperature and rate, crosslinking conditions (e.g., polymer concentration), and the type and molecular weight of the polymer used.

[0059] In some embodiments, the scaffold materials and compositions disclosed herein are not seeded or otherwise loaded with cells prior to their application to or implantation in a subject. Alternatively, in other embodiments, the scaffold materials and compositions disclosed herein are seeded or otherwise loaded with cells (e.g., HSCs) prior to their application to or implantation in a subject. In other embodiments, the scaffold material is selected from: polylactic acid, polyglycolic acid, PLGA polymers, alginate and alginate derivatives, polycaprolactone, calcium phosphate-based materials, gelatin, collagen, fibroin, hyaluronic acid, laminin-rich gels, agarose, natural and synthetic polysaccharides, polyamino acids, peptides, polyesters, polyanhydrides, polyphosphazene, poly(vinyl alcohol), poly(epoxyalkylene), poly(allylamine) (PAM), poly(acrylate), modified styrene polymers, pululanic polyols, poloxamer, poly(uronic acid), poly(vinylpyrrolidone), and any combination or copolymer thereof. Other exemplary scaffold materials, compositions, and methods of using and preparing them are described in U.S. Patent Publications 2008 / 0044900, 2013 / 0331343, 2015 / 0366956, 2014 / 0112990, 2014 / 0227327, and 2015 / 0359928, which are incorporated herein by reference in their entirety.

[0060] The scaffold materials disclosed herein can be further modified, for example, by including compounds or excipients to impart osteoinductive properties (e.g., calcium phosphate). Similarly, compounds or excipients can be included in the scaffold materials to influence their mechanical properties. For example, to modulate the mechanical properties of the scaffold materials, polymers such as hard polycaprolactone (PCL) and soft polyethylene glycol (PEG) can be used in combination with alginate.

[0061] Scaffold materials can be used to control the in vivo presentation or release of one or more growth factors, differentiation factors, and / or homing factors, for example, after application or implantation of the scaffold material or composition. For example, an EDC / NHS chemistry approach can be used, employing carboxylic acid groups on an alginate backbone to bind DLL-4 to the scaffold material. This presentation or release of one or more growth factors, differentiation factors, and / or homing factors can be achieved by encapsulating or coupling (e.g., covalently binding or coupling) these molecules within or on the scaffold material (e.g., coupling the molecules to the alginate backbone). The spatial and temporal presentation of these molecules can be precisely controlled by fine-tuning the chemical reactions used to couple these molecules and by selecting or altering the physical and chemical properties of the scaffold material. Therefore, these scaffold materials are particularly useful for controlling the in vivo delivery and / or presentation of one or more molecules (e.g., growth factors) that can be encapsulated within or coupled to the scaffold material. Thus, based on the selection of one or more growth factors, differentiation factors, and / or homing factors, the release of these molecules from the scaffold material can be optimized to achieve and precisely control the behavior (e.g., proliferation, migration, and / or differentiation) of transplanted stem cells and progenitor cells in vivo.

[0062] In some respects, upon application or implantation of the compositions disclosed herein, one or more growth factors contained in such compositions promote the formation of tissue (e.g., bone tissue) on or around the applied composition, thereby forming nodules that serve as ectopic bone marrow nests. As used herein, the term "growth factor" generally refers to any bioactive agent, polypeptide, hormone, cytokine, chemokine, or compound that causes or otherwise promotes cell proliferation, and in some respects includes osteogenic growth factors and their analogues. Exemplary growth factors include bone morphogenetic proteins such as BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, and BMP-14. One or more growth factors may be encapsulated in a scaffold material and released from the scaffold material over an extended period of time (e.g., about 7-14 days). One or more growth factors may be encapsulated in a scaffold material and released from the scaffold material over about 1-50, 5-25, 10-20, or 17-18 days. In some implementations, one or more growth factors (e.g., BMP-2) are released over a sufficient period of time to allow tissue (e.g., bone tissue) to form on or around the implanted composition, thus forming nodules.

[0063] The compositions disclosed herein also comprise one or more differentiation factors. As used herein, the term "differentiation factor" refers to any molecule that promotes cell differentiation. For example, any differentiation factor that promotes the differentiation of stem cells or progenitor cells into one or more lymphoid lineage cells. In some aspects, differentiation factors promote the migration of transplanted cells (e.g., HSCs) to one or more secondary sites, such as the thymus and / or lymph nodes, where these cells then mature. For example, transplanted stem cells may home to or otherwise infiltrate a nodule, where these cells come into contact with differentiation factors (e.g., Notch ligands, such as DLL-2 and / or DLL-4), and then further migrate to the thymus, where they will mature into T cells. In some aspects, differentiation factors promote the differentiation and maturation of transplanted cells within a nodule. For example, transplanted stem cells may home to or otherwise infiltrate a nodule, where these cells come into contact with differentiation factors (e.g., IL-7 or IL-15), and then differentiate into mature B cells within the nodule.

[0064] In some respects, the differentiation factors disclosed herein can be modified as needed to, for example, optimize the performance of the compositions disclosed herein. In some embodiments, a Notch-binding peptide fragment of DLL-1 or DLL-4 (approximately 2 kDa) can be used instead of the complete protein (approximately 70 kDa) to allow for greater functionalization of the alginate scaffold material. In some embodiments, to further enhance the effectiveness of the scaffold material, angiogenic vascular endothelial growth factor (VEGF) and / or chemokinetic SDF-1 can be incorporated.

[0065] In some embodiments, the compositions disclosed herein also comprise one or more “homing factors,” a term generally referring to any composition that promotes the migration or homing of transplanted stem cells to nodules formed after administration or implantation of the compositions disclosed herein. Stem cell transplantation is likely due to the innate ability of hematopoietic stem cells to “hom” from the bloodstream to the bone marrow after infusion into a subject. Homing of transplanted stem cells (e.g., HSCs) to nodules is enhanced by incorporating, encapsulating, or tethering one or more homing factors into a scaffold material containing the compositions disclosed herein. For example, the methods and compositions disclosed herein can be used to improve (e.g., increase) the ability of infused HSCs to hom to ectopic bone marrow nests formed by nodules from the tissue (e.g., blood) infused with these HSCs during transplantation. As used herein, the term “homing” refers to the migration, movement, or other concentration of transplanted stem cells (e.g., HSCs or progenitor cells) from a first specific tissue or region (e.g., blood) in which they were infused to a second tissue or region where they are needed (e.g., a nodule formed in a subject after implantation of the composition). Various factors within endogenous stem cell nests regulate the homing of HSCs to bone marrow stem cell nests, and one or more of these factors can be incorporated into the compositions disclosed herein. One such factor that promotes the retention and homing of HSCs within endogenous bone marrow is stem cell differentiation factor (SDF-1). Therefore, in some aspects, the compositions disclosed herein contain SDF-1, which promotes the homing of transplanted stem cells to nodules and the subsequent implantation of these cells into ectopic bone marrow nests formed from such nodules.

[0066] The methods and compositions disclosed herein can be used for stem cell transplantation (e.g., HSC transplantation). As used herein, the term "hematopoietic stem cell" or "HSC" refers to stem cells capable of differentiating into hematopoietic lineages and producing all blood cell types, such as white blood cells and red blood cells, including bone marrow lineages (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (e.g., T cells, B cells, NK cells). Stem cells are defined by their ability to form multiple cell types (pluripotency) and their capacity for self-renewal. Hematopoietic stem cells can be identified, for example, by cell surface markers such as CD34-, CD133+, CD48-, CD150+, CD244-, cKit+, Sca1+ and the absence of lineage markers (especially negative for B220, CD3, CD4, CD8, Mac1, Gr1, and Ter119).

[0067] As used herein, the term "progenitor cell" encompasses pluripotent cells directed toward the hematopoietic lineage, which typically do not self-renew and are capable of differentiating into several cell types of the hematopoietic system, such as granulocytes, monocytes, erythrocytes, megakaryocytes, B cells, and T cells. These progenitor cells include, but are not limited to, short-term hematopoietic stem cells (ST-HSCs), pluripotent progenitor cells (MPPs), common bone marrow progenitor cells (CMPs), granulocyte-monocyte progenitor cells (GMPs), megakaryocyte-erythrocyte progenitor cells (MEPs), and directed lymphocyte progenitor cells (CLPs). The presence of hematopoietic progenitor cells can be functionally determined as colony-forming unit cells (CFU-Cs) in a complete methylcellulose assay, or phenotypically determined by detecting cell surface markers (e.g., CD45-, CD34+, Ter119-, CD16 / 32, CD127, cKit, Sca1) using analyses known to those skilled in the art.

[0068] Some aspects of the methods disclosed herein involve administering or otherwise transplanting stem cells to a subject in need, such that the administered stem cells are implanted into an ectopic bone marrow nest of a nodule (e.g., a bone nodule) formed after administration of the compositions disclosed herein to the recipient subject. As used herein, "implantation" of stem cells includes hematopoietic stem cells, meaning the placement of stem cells, for example, in an animal via injection, where the stem cells persist in vivo. This can be readily measured by the capacity of the stem cells, such as their ability to promote ongoing immune cell and / or blood cell formation. Successful stem cell transplantation depends on the ability to implant a sufficient quantity of transplanted stem cells into the subject's tissues; therefore, the compositions and methods disclosed herein increase the number or volume of sites and tissues where these transplanted stem cells can be implanted.

[0069] As used herein, the term "application" generally refers to placing the compositions described herein into a subject by means of methods or pathways that cause transplanted stem cells to migrate or home to ectopic bone marrow nests of the nodule (e.g., placing these compositions parenterally or implanting them into the subject). In some aspects, such as prior to stem cell transplantation, the compositions disclosed herein are applied to or implanted into multiple sites in the subject. In some embodiments, the compositions disclosed herein are applied to sites near the subject's lymphatic system (e.g., near one or more of the subject's neck, groin, and armpit). In some aspects, the nodules disclosed herein are removed (e.g., by surgical excision) after the subject's immune system has been rebuilt.

[0070] In some embodiments, the compositions and methods disclosed herein can be used to treat any condition, disease, symptom, or complication requiring transplantation of hematopoietic stem cells and / or progenitor cells. Examples of such conditions include hematologic malignancies and non-malignant hematologic diseases. The compositions and methods disclosed herein can also be used to reconstruct T cells and B cells, and therefore can be broadly applied to other diseases involving immunodeficiency, such as age-related vaccine failure, autoimmune diseases (e.g., rheumatoid arthritis and diabetes), infectious diseases, etc.

[0071] As used herein, the term "subject" refers to any person or animal. In some respects, an animal is a vertebrate, such as a primate, rodent, domesticated animal, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Domesticated and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, canine species such as dogs, foxes, wolves, bird species such as chickens, emus, ostriches, and fish such as trout, catfish, and salmon. A patient or subject includes any subset of the foregoing categories, such as all of the foregoing categories but not including one or more groups or species such as humans, primates, or rodents. In some embodiments, a subject is a mammal (e.g., a primate or a human). In some embodiments, a mammal is a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cattle, and is not limited to these examples. Mammals other than humans can be advantageously used as subjects, for example, as animal models representing hematologic malignancies. Additionally, the methods described herein can be used to treat domesticated animals and / or pets. Subjects can be male or female.

[0072] In some embodiments, a subject may be someone who has been previously diagnosed or otherwise identified as having or suffering from a condition, disease, or stem cell disorder. A "subject" who "needs" treatment for a specific condition (e.g., stem cell disorder) may be a subject who has the condition, is diagnosed with the condition, or has an increased risk of developing the condition relative to a given reference population. In some embodiments, the treatment methods described herein include selecting subjects who are diagnosed with, suspected of having, or at risk of developing a hematologic malignancy or immunodeficiency. In some embodiments, the methods described herein include selecting subjects who are diagnosed with, suspected of having, or at risk of developing a non-malignant disease as described herein.

[0073] In some embodiments, the methods disclosed herein accelerate the reconstruction of a subject's immune system from endogenous progenitor cells (HSCs). In some embodiments, the methods disclosed herein accelerate reconstruction from endogenous progenitor cells (HSCs) by approximately 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, or 4 orders of magnitude or more. In some embodiments, the methods disclosed herein accelerate reconstruction from endogenous progenitor cells (HSCs) by more than 2 orders of magnitude.

[0074] In some embodiments, the methods disclosed herein accelerate the reconstruction of a subject's immune system from transplanted progenitor cells (HSCs). In some embodiments, the methods disclosed herein accelerate the reconstruction from transplanted progenitor cells (HSCs) by about 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, or 4 orders of magnitude or more. In some embodiments, the methods disclosed herein accelerate the reconstruction from transplanted progenitor cells (HSCs) by more than 2 orders of magnitude.

[0075] It should be understood that the application of this invention is not limited to the details set forth or exemplified in the specification. This invention encompasses other embodiments and can be practiced or carried out in various ways. Furthermore, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered limiting.

[0076] Although certain reagents, compounds, compositions, and methods of the present invention have been specifically described according to some embodiments, the following examples are only for illustrating the methods and compositions of the present invention and are not intended to limit the present invention.

[0077] Unless explicitly indicated to the contrary, the quantifier “a” used in this specification and claims shall be understood to include multiple indicators. Unless the context indicates or clearly indicates otherwise, a claim or description including “or” among one or more members of the group is considered to satisfy if one, more than one, or all of the group members are present in, used in, or otherwise associated with a given product or process. The invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise associated with a given product or process. The invention also includes embodiments in which more than one or all of the group members are present in, used in, or otherwise associated with a given product or process. Furthermore, it should be understood that, unless otherwise indicated or unless a contradiction or inconsistency would be apparent to a person skilled in the art, the invention covers all variations, combinations, and modifications in which one or more limitations, elements, clauses, descriptive terms, etc., of one or more of the listed claims are incorporated into another claim (or any other related claim) dependent on the same basic claim. When elements are presented in a list format (e.g., in Markush groups or a similar format), it should be understood that each subgroup of elements is also disclosed, and any element can be removed from the group. It should be understood that, generally, when an aspect of the invention is referred to as containing a particular element, feature, etc., certain embodiments or aspects of the invention consist of or are substantially composed of such elements, features, etc. For the purpose of simplicity, these embodiments will not be described in such detail in each case herein. It should also be understood that any embodiment or aspect of the invention may be expressly excluded from the claims, whether or not a specific exclusion is described in this specification. Publications and other references mentioned herein to describe the background of the invention and to provide further details about its practice are incorporated herein by reference.

[0078] Example

[0079] Example 1 - Synthetic materials for inducing osteophyte formation

[0080] The inventors used alginate, a natural anionic polysaccharide, as a hydrogel scaffold material. Furthermore, alginate is suitable for incorporating the cationic effective bone morphogenetic growth factor, bone morphogenetic protein-2 (BMP-2), to generate bone nodules, such as… Figures 2A-2E The performance of BMP-2 growth factor loading into and releasing from alginate was optimized (e.g., total amount at injection and cross-linking density of alginate to control release), with the aim of generating functional active bone nodules within 1–2 weeks after subcutaneous injection in mice.

[0081] Both T cells and B cells originate from specific types of HSC progenitor cells, namely common lymphoprogenitor cells (CLPs). Within the bone marrow, the Notch pathway is crucial for lymphocyte specialization. CLPs exposed to Notch ligands delta-like ligands 1 or 4 (DLL-1 / 4) migrate to the thymus and differentiate into T cells. CLPs exposed to interleukin-7 (IL-7) in the bone marrow differentiate into B cells. To concretize lymphocyte differentiation in transplanted HSCs homing to ectopic bone marrow, the inventors evaluated the effect of incorporating the Notch ligand DLL-4 into the ectopic bone marrow. Using EDC / NHS chemistry, DLL-4 is bound to the carboxylic acid group on the alginate backbone to guide T cell differentiation in HSCs.

[0082] The in vitro differentiation potential of hydrogels containing growth factor BMP-2 and differentiation factor DLL-4 was tested using Lin-c-kit+Sca-1+ (LKS) cells—pending hematopoietic stem cells and progenitor cells. The inventors analyzed differentiation into CD4+ and CD8+ T cells and compared different strategies for incorporating DLL-4. Figure 3A ) It was observed that DLL-4 binding and tethering are essential for biological activity. Next, the inventors tested a subset of gels that performed best in sublethal irradiated mice, and as shown... Figure 3B The study showed that the rate of T cell and B cell production was accelerated compared to the untreated group.

[0083] In addition to the above studies, the inventors homogenized and sectioned the obtained ectopic bone marrow nodules to determine the cell population. The levels of stromal cell-derived factor-1 (SDF-1) chemotaxis in HSCs were also measured, and the histological assessment of the vascular distribution and cellular composition of the ectopic bone marrow nodules was compared with that of healthy bone marrow. The inventors will use a specific molar ratio (e.g., 10) within a certain range. -9 Up to 10 -6 The system was further optimized by combining DLL-4 with an alginate backbone. Furthermore, to specifically drive B cell production in the bone marrow, the inventors incorporated a dose range (100-500 ng) of soluble IL-7 instead of DLL-4 into the hydrogel, with the aim of sustained release of soluble IL-7 for at least one week. Flow cytometry was used to track lymphoprogenitor cells as they differentiated, matured, and migrated through the bone marrow, peripheral lymphoid organs, blood, and spleen.

[0084] Example 2 - Characterizing lymphocyte remodeling and immune repositories after HSTC

[0085] Primary T cells and B cells are essential for a continuous response to unfamiliar pathogens via cell-mediated immunity and humoral immunity, respectively. The regeneration of primary lymphocytes from donor cells after HSCT was examined. Bone marrow was harvested from donor mice, and bone marrow with T-cell and B-cell anemia (>95%) was transplanted into lethally irradiated homologous recipient mice, and the regeneration of T-cell and B-cells was tracked, as shown in the following figures. Figure 4A As explained, TCR diversity was determined by sequencing the T cell receptor (TCR) of CD3+ cells and examining the frequency and distribution of the variable region (V) and linker region (J) of the TCR gene. Figure 4B As explained.

[0086] The inventors will simulate a transplantation procedure performed in a clinical setting and examine the transplanted cells using whole bone marrow or purified LKS cells derived from bone marrow. The best-performing hydrogel scaffold material identified in the study described in Example 1 will be tested along with the transplanted cells. Flow cytometry and complete blood counts will be used to analyze T cells and B cells in the hematopoietic compartment. Additionally, the inventors will use T cell receptor excision loop (TREC) analysis to measure the output of primary T cells from the thymus and assess the primary CD3+CD4+ / CD8+CD62L ratio. hi Similarly, the inventors will assess the reconstitution of primary B cells (B220+CD40+CD84+) and the B cell receptor repertoire in the bone marrow.

[0087] Example 3: Macroporous hydrogel with DLL-4, SDF-1, and BMP-2

[0088] Methacrylate-functionalized sodium alginate (ProNova Biomedical) was used to prepare methacrylated alginate (MA-Alg). 1–10 nmol of delta-like ligand 4 (DLL-4, R&D systems) was conjugated to MA-Alg using EDC-NHS coupling. An injectable macroporous hydrogel was synthesized by low-temperature redox-induced free radical polymerization of MA-alginate and 4-arm methacrylated polyethylene glycol (MA-PEG), yielding a 2.5 wt% hydrogel. Bone morphogenetic protein-2 (BMP-2, R&D systems) and stem cell differentiation factor-1 (SDF-1, R&D systems) were added to the mixture, followed by cold polymerization. Lin isolated from bone marrow was used... - c-kit + Sca-1 + (LKS) cells convert to CD4 + and CD8 + T cell differentiation was used to assess DLL-4 bioactivity. Sublethal and lethal irradiation in C57BL / 6 mice were used to simulate immunodeficiency and myeloablative transplant conditioning, respectively. (The last sentence appears to be incomplete and unrelated to the preceding text.) +Transplantation was performed in mouse strains, and the genetic variation of CD45.1 was monitored. + Cells were generated from B6.SJL mouse donors. Hydrogels were injected subcutaneously, and the generation of bone nodules and the associated hematopoietic environment were monitored using micro-computed tomography (μCT) and histology, respectively. Recovery of immune cells in peripheral blood was monitored periodically using FACS analysis. T-cell receptor (TCR) sequencing was used to determine the diversity of the T-cell repertoire.

[0089] In the MA-Alg / MA-PEG hydrogel, DLL-4 remained tethered to the scaffold, while BMP-2 and SDF-1 were co-released over a period of 2.5 weeks. In vitro, the hydrogel-tethered DLL-4 released Lin... - c-kit + Sca-1 + (LKS) cells differentiate into primary CD4 cells. + and CD8 + Cells, and equivalent to native DLL-4 absorbed on TCPS. In vivo, the scaffold induced the formation of subcutaneous bone nodules in mice. Histological analysis of the injected hydrogel showed the formation of bone nodules with bone marrow, which were filled with lymphoprogenitor cells within 2 weeks post-injection. In sublethal irradiated mice, the BMP-2 / DLL-4 hydrogel accelerated the reconstitution of T-lymphocytes and B-lymphocytes by more than two orders of magnitude within 3 weeks by recruiting and driving lymphocyte differentiation of endogenous progenitor cells (HSCs). In transplanted lethal irradiated mice, the hydrogel acted as a preferred site for the engraftment of transplanted progenitor cells (HSCs) and induced their differentiation into T cells and B cells. The recovery of the adaptive immune system followed a similar trend as in sublethal irradiated mice. Hydrogel-mediated expansion of the T-competent progenitor cell pool increased thymic output in transplanted mice and corresponded to an increase in the primary T-cell pool. Sequencing of TCRs revealed increased frequency and diversity, as measured by recombination of the variable region (V) and linker region (J) of the TCR gene in hydrogel-mediated T cell repertoires.

[0090] The results indicate that programmable biomaterials with biological cues can remodel various aspects of the bone marrow matrix. By increasing the available sites for donor cell implantation and providing cues for lymphocyte proliferation, the remodeling of adaptive immunity after HSCT can be accelerated, potentially reducing HSCT-related immune complications and improving the treatment of hematologic disorders.

Claims

1. A composition comprising: Porous implantable scaffold materials Growth factors containing BMP-2, and Differentiation factors including δ-like 4, and One or more homing factors can be selected. The differentiation factor is covalently bound to the scaffold material or to a lineage covalently bound to the scaffold material, and promotes the differentiation of hematopoietic stem cells into lymphoid lineage cells. The growth factor is encapsulated in the scaffold material and the amount of the growth factor effectively promotes ectopic bone tissue formation on or around the applied scaffold material to form ectopic bone marrow nodules for the infiltration and / or implantation of hematopoietic stem cells.

2. The composition of claim 1, wherein the scaffold material is a hydrogel.

3. The composition of claim 1 or 2, wherein the scaffold material comprises alginate.

4. The composition of claim 1 or 2, wherein the scaffold material comprises anionic alginate.

5. The composition of claim 1 or 2, wherein the scaffold material is selected from: polylactic acid, polyglycolic acid, PLGA polymer, polycaprolactone, calcium phosphate-based materials, gelatin, collagen, fibroin, laminin-rich gel, natural polysaccharides, synthetic polysaccharides, polypeptides, polyanhydrides, polyphosphazene, poly(vinyl alcohol), poly(epoxyalkylene), poly(allylamine) (PAM), poly(acrylate), modified styrene polymer, poloxamer, poly(vinylpyrrolidone), and any combination or copolymer thereof.

6. The composition of claim 1 or 2, wherein the scaffold material is selected from: alginate, alginate derivatives, hyaluronic acid, agarose, polyamino acids, polyester, and any combination thereof.

7. The composition of claim 1 or 2, wherein the scaffold material is poly(uronic acid).

8. The composition of claim 1 or 2, wherein the growth factor is released from the scaffold material within 7-30 days.

9. The composition of claim 1 or 2, wherein one or more of the homing factors comprise stem cell differentiation factors.

10. The composition of claim 1 or 2, wherein one or more of the homing factors are encapsulated in the scaffold material.

11. The composition of claim 1 or 2, wherein one or more of the homing factors are released from the scaffold material within 7-30 days.

12. Use of a composition comprising a scaffold material in the preparation of a medicament for assisting in the reconstruction of the immune system of a subject in need, said composition comprising: Porous implantable scaffold materials Growth factors containing BMP-2, and Differentiation factors including δ-like 4, and One or more homing factors can be selected. The differentiation factor is covalently bound to the scaffold material or to a lineage covalently bound to the scaffold material, and promotes the differentiation of hematopoietic stem cells into lymphoid lineage cells. The growth factor is encapsulated in the scaffold material and the amount of the growth factor effectively promotes ectopic bone tissue formation on or around the applied scaffold material to form ectopic bone marrow nodules for the infiltration and / or implantation of hematopoietic stem cells.

13. Use of a composition comprising a scaffold material in the preparation of a medicament for forming ectopic hematopoietic stem cell nests in subjects of need, said composition comprising: Porous implantable scaffold materials Growth factors containing BMP-2, and Differentiation factors including δ-like 4, and One or more homing factors can be selected. The differentiation factor is covalently bound to the scaffold material or to a lineage covalently bound to the scaffold material, and promotes the differentiation of hematopoietic stem cells into lymphoid lineage cells. The growth factor is encapsulated in the scaffold material and the amount of the growth factor effectively promotes ectopic bone tissue formation on or around the applied scaffold material to form ectopic bone marrow nodules for the infiltration and / or implantation of hematopoietic stem cells.

14. Use of a composition comprising a scaffold material in the preparation of a medicament for improving the implantation of transplanted hematopoietic stem cells into stem cell nests in a subject in need, said composition comprising: Porous implantable scaffold materials Growth factors containing BMP-2, and Differentiation factors including δ-like 4, and One or more homing factors can be selected. The differentiation factor is covalently bound to the scaffold material or to a lineage covalently bound to the scaffold material, and promotes the differentiation of hematopoietic stem cells into lymphoid lineage cells. The growth factor is encapsulated in the scaffold material and the amount of the growth factor effectively promotes ectopic bone tissue formation on or around the applied scaffold material to form ectopic bone marrow nodules for the infiltration and / or implantation of hematopoietic stem cells.

15. Use of a composition comprising a scaffold material in the preparation of a medicament for increasing the implantation site of transplanted stem cells in a subject in need, said composition comprising: Porous implantable scaffold materials Growth factors containing BMP-2, and Differentiation factors including δ-like 4, and One or more homing factors can be selected. The differentiation factor is covalently bound to the scaffold material or to a lineage covalently bound to the scaffold material, and promotes the differentiation of hematopoietic stem cells into lymphoid lineage cells. The growth factor is encapsulated in the scaffold material and the amount of the growth factor effectively promotes ectopic bone tissue formation on or around the applied scaffold material to form ectopic bone marrow nodules for the infiltration and / or implantation of hematopoietic stem cells.

16. The use of any one of claims 12 to 15, wherein the scaffold material comprises a hydrogel material.

17. The use of any one of claims 12 to 15, wherein the scaffold material comprises alginate.

18. The use of any one of claims 12 to 15, wherein the scaffold material comprises anionic alginate.

19. The use of any one of claims 12 to 15, wherein the scaffold material is selected from: polylactic acid, polyglycolic acid, PLGA polymer, polycaprolactone, calcium phosphate-based materials, gelatin, collagen, fibroin, laminin-rich gels, natural polysaccharides, synthetic polysaccharides, polypeptides, polyanhydrides, polyphosphazene, poly(vinyl alcohol), poly(epoxyalkylene), poly(allylamine) (PAM), poly(acrylate), modified styrene polymers, poloxamer, poly(vinylpyrrolidone), and any combination or copolymer thereof.

20. The use according to any one of claims 12 to 15, wherein the scaffold material is selected from: alginate, alginate derivatives, hyaluronic acid, agarose, polyamino acids, polyester, and any combination thereof.

21. The use according to any one of claims 12 to 15, wherein the scaffold material is poly(uronic acid).

22. The use according to any one of claims 12 to 15, wherein the growth factor is released from the scaffold material within 7 to 30 days.

23. The use according to any one of claims 12 to 15, wherein one or more of the homing factors comprise stem cell differentiation factors.

24. The use of any one of claims 12 to 15, wherein one or more of the homing factors are encapsulated in the scaffold material.

25. The use of any one of claims 12 to 15, wherein one or more of the homing factors are released from the scaffold material within 7 to 30 days.

26. The use according to any one of claims 12 to 15, wherein the differentiation factor promotes the proliferation of lymphocytes in the transplanted stem cells.

27. The use according to any one of claims 12 to 15, wherein the subject has undergone stem cell transplantation.

28. The use according to any one of claims 12 to 15, wherein the subject is immunocompromised.

29. The use of any one of claims 12 to 15, wherein one or more of the said lymphoid and bone marrow lineage cells are CD4+, CD8+, and Mac-1+ / GR-1+.

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