Skeletal and hematopoietic micro-environment

Abstract

The present invention relates to a ground extracellular matrix (ECM) produced by a human stem cell line with chondrogenic activity, wherein the human stem cell line is genetically modified to overexpress Bone Morphogenetic Protein-2 (BMP-2). The invention further relates to a composition comprising said extracellular matrix, and a gel-like substrate or precursor thereof. The invention further relates to a kit comprising said composition. The invention further relates to a method of manufacturing said composition. The invention further relates to a method for bone and / or bone marrow formation in vivo. The invention further relates to a method for testing a candidate pharmaceutical product. The invention further relates to a method for identifying treatment responders. The invention further relates to said composition for use in medicine.

Description

[0001]P7250PC001 Skeletal and hematopoietic micro-environment Technical field The present invention relates to a composition comprising ground extracellular matrixand a gel-like substrate or precursor thereof, its manufacturing and its use for formationof forming bone and / or bone marrow, testing candidate drugs or identifying treatment responders. Background Bone is a dynamic and highly specialized tissue that provides structural body support and organ protection, while housing the bone marrow compartment, responsible for life-long hematopoiesis—the production of blood cells. Bone forms and regeneratesthrough a canonical pathway defined as endochondral ossification. This processinvolves the condensation and differentiation of mesenchymal stem cells (MSCs) into chondrocytes, leading to the formation of a cartilage template. This cartilage tissue is transient, as upon further vascularization and remodeling a fully mature bone and bone marrow compartment is being formed. Most of the knowledge on the endochondral ossification process has been derived from mouse studies, but much remain to be discovered on the cellular and molecular mechanisms governing human bone formation. In this process, MSCs are essential as being at the origin of the transient cartilage tissue, but also persisting in the final bone and bone marrow compartments. MSCs are multipotent stem cells that can differentiate into chondrocytes, adipocytes, osteoblasts (bone-forming cells), osteocytes (mature bone cells), but also stromal cells that actively support bone homeostasis as well as the hematopoietic activity. If fundamental knowledge is lacking on the human bone developmental / formation process, the same holds true for bone disorders. In fact, the vast majority of existingcancers emerge or ultimately develop in the bones, non-exhaustively includingleukemia, breast, prostate, bladder cancers or neuroblastoma. The bones are thusconsidered a privileged “harbor” for cancer cells, and this is also associated with a very poor survival prognostic: bone-developing cancers account for ~3 million deaths each year. Research studies pinpoint the essential role of MSCs (and derived populations) in the bone in cancer initiation, progression and relapse mechanisms. Most importantly, MSCs have been involved in mechanisms of treatment resistance, through cancer stem cell protection. Overall, there is a clear need for robust models that can faithfullyP7250PC002 mimic human bone biology in order to advance our understanding of bone-developing cancers and develop new treatments. In vitro and in vivo tools can be exploited to understand better how human bone tissuescan form and function in homeostasis and disease contexts. However, major limitationsarise from existing approaches, including a lack of biological relevance in reproducibility. For instance, the ex vivo expansion and maintenance of bone marrowderived MSCs (BM-MSCs) functionality is limited. Moreover, BM-MSCs can be veryheterogenous cellular populations, and substantial donor-to-donor variability exists. This has largely challenged their robust exploitation in in vitro and in vivo models. Thus,developing new technologies that can lead to a more standardized differentiation ofhuman cells -including BM-MSC3- will be essential in order to design and exploitrelevant models of the human bone tissues.SummaryThe inventors of the present disclosure have identified a composition comprising aground extracellular matrix (ECM) produced by a human stem cell line and a gel-likesubstrate (hydrogel) or precursor thereof, that can be used to produce bone and bonemarrow in a reproducible and easy-to-handle fashion. The disclosed composition offersthe production of batches of material with constant properties, which enhancesreproducibility. Importantly, the ground ECM can be used for cartilage, fat, bone andbone marrow formation upon mixture with a gel like substrate (hydrogel) or precursorthereof. The disclosed composition results in a protein-based matrix of human originwith intrinsic osteoinductive properties that are constitutively active eliminating the needfor exogenous trophic factor supplementation, which is commonly required inconventional methods. The disclosed composition enables the efficient andreproducible generation of human bone organoids, used to model samples fromleukemia patients, which are typically challenging to model using current standard preclinical systems. In addition to applications in blood cancers, the technology shows strong potential in the modelling of bone-metastasizing cancers, where effective animalmodels are limited and patient survival rates remain critically low. Most importantly, thedisclosed composition is injectable, thus having the advantage of being injected into a desired site, which allows for targeted delivery and minimally invasive procedures. Theinventors have developed a hydrogel-extracellular matrix reagent to be used for the invitro and in vivo culture of human cells. Precisely, the reagent confers standardizedP7250PC003human cell differentiation both in vitro and in vivo, thus facilitating the formation oftissues constitutive of human bones, including cartilage, fat, bone and bone marrowtowards the modelling of their various functions. This model system offers a powerfultool for studying fundamental human skeletal biology and disease, providing astandardized environment to investigate the cellular and molecular mechanisms oftissue formation, the role of the bone marrow niche in health and disease, and thepotential for personalized therapeutic approaches. Further, it offers a reproducible andeasy-to-handle set-up for determining the efficacy of drugs or for identifying treatmentresponders. The platform's versatility and compatibility with patient-derived materialsupports the creation of personalized bone organoids for disease modelling and therapeutic testing, aligning with precision medicine strategies. Furthermore, the minimally invasive gel formulation, combined with the scalable and sustainable production process, enables the formation of up to six human mini-bone organoids perresearch animal reducing animal use by at least 75% per study, and significantlylowering labour and time requirements.In one aspect the invention relates to a ground extracellular matrix (ECM) produced bya human stem cell line.In one aspect the invention relates to a ground extracellular matrix (ECM) produced bya human stem cell line with chondrogenic activity, wherein the human stem cell line is genetically modified to overexpress Bone Morphogenetic Protein-2 (BMP-2).In a further aspect the invention relates to an injectable composition comprising:a. the ground extracellular matrix (ECM) as defined in the presentdisclosure, and b. a hydrogel or precursor thereof.In another aspect the invention relates to a kit comprising the composition as defined inthe present disclosure, phosphate-buffered saline, cell culture media, and instructions for use.In another aspect the invention relates to a method of manufacturing the compositionas defined in the present disclosure, the method comprising the following steps: a. culturing a stem cell line in vitro;P7250PC004 b. exposing the stem cell line to differentiation factors, therebydifferentiating said stem cell line into mesenchymal lineage cell types;c. culturing the differentiated cells of step b, thereby producing anextracellular matrix (ECM); d. lyophilizing the cells comprised in the ECM of step c;e. grinding the ECM of step d, thereby obtaining a ground ECM;and / or f. mixing the ground ECM with a hydrogel or precursor thereof.In another aspect the invention relates to an in vivo method for bone formation, the method comprising the following steps: a. administering the composition as defined in the present disclosure to asubject; and b. maintaining for at least 1 week, such as 2 weeks, such as 3 weeks.In another aspect the invention relates to an in vivo method for bone and bone marrowtissue formation, the method comprising the following steps: a. administering the composition as defined in the present disclosure to asubject; and b. maintaining for at least 2 weeks, such as 3 weeks, such as 4 weeks,such as 5 weeks, such as 6 weeks, such as 7 weeks, such as 8 weeks.In another aspect the invention relates to an in vitro method for bone and bone marrowtissue formation, the method comprising the following steps: a. placing the composition as defined in the present disclosure in culturemedia; b. administering cells;c. incubating for at least 1 week, such as 2 weeks, such as 3 weeks, suchas 4 weeks, such as 5 weeks, such as 6 weeks, such as 7 weeks, such as 8 weeks. In another aspect the invention relates to a method for testing a candidate pharmaceutical product, the method comprising the following steps:P7250PC005 a. establishing bone and bone marrow according to the methods asdisclosed herein; b. administering pathological cells, wherein the pathological cells arepatient-derived pathological cells or genetically engineered pathologicalcells, to the bone and bone marrow of step a, thereby establishing amodel pathological bone and bone marrow;c. assessing the viability and / or cell number of the pathological cells;d. administering a candidate pharmaceutical product;e. assessing the viability and / or cell number of the pathological cells;and f. classifying the candidate pharmaceutical product as effective againstpathological cells if the viability and / or cell number is decreased in step e compared to step c. In another aspect the invention relates to a method for identifying treatmentresponders, the method comprising the following steps:a. obtaining cells from a patient, wherein the cells are primary cancer cellsand their premalignant state cells, hematopoietic cells from patients with genetic disorders, marrow failure, or clonal hematopoiesis, tumor- initiating cells or cancer stem cells, and / or inflammatory or immune cells from autoimmune bone pathologies; b. mixing the cells of step a. with the composition as defined in the presentdisclosure; c. administering the mixture of step b. to a subject;d. maintaining the mixture in the subject for at least 2 weeks, such as 3weeks, 4 weeks, such as 5 weeks, such as 6 weeks, such as 7 weeks, such as 8 weeks, thereby establishing a model of the bone and bone marrow of said patient; e. assessing the viability and / or cell number of the administered cells in thebone marrow; f. administering a pharmaceutical product;g. assessing the viability and / or cell number of the administered cells;andP7250PC006 h. classifying the patient as a responder to the pharmaceutical product ifthe viability and / or cell number is decreased in step g compared to stepe.In another aspect the invention relates to a product obtained by the methods asdisclosed herein. Description of DrawingsFigure 1: Development and Characterization of OssiGel: A Custom Hydrogel forEctopic Humanized Bone and Bone Marrow Formation in Nude Mice for CancerResearch. A) Production process of the invention defined as “OssiGel”. A human cellsource – in particular the Mesenchymal Sword of Damocles – BMP2 (MSOD-B) cellline described in Pigeot et al. Advanced Materials 2021 (1) – is seeded on 3D porousscaffolds, particularly collagen scaffolds. The cells on the scaffolds are exposed todifferentiation factors – in particular to chondrogenic factors - for 3 weeks, for theformation of mature tissues. After 3 weeks of in vitro differentiation, the resulting humancartilage tissue is lyophilized. The lyophilized cartilage tissue is further ground in orderto obtain a fine extracellular matrix (ECM) powder. The ground, lyophilized ECM ismixed with a natural or synthetic hydrogel, in particular fibrin and thrombin that can beinjected. B) Overview of the exploitation of OssiGel in vivo. C) Overview of theexploitation of OssiGel in vitro.Figure 2: The particles of the lyophilized, ground extracellular matrix is a finepowder consisting of small particles. A) The lyophilized cartilage. B) Overview of theground lyophilized extracellular matrix (ECM). C) scanning electron microscopy (SEM)micrograph of ground ECM. Scale bar 100 µm. D) Particle size distribution. The datarepresent Means ^ standard deviation (SD) on a logarithmic scale. Small particles havea mean size of 6.7 ^ 14.5 micrometers and big particles have a mean size of 272 ^ 226micrometers.Figure 3: The amount of glycosaminoglycan in the lyophilized cartilage ismaintained during and after the grinding process. Glycosaminoglycans (GAGs)retention during and after the grinding process of the lyophilized extracellular matrix.P7250PC007Figure 4: The lyophilized and ground ECM can be successfully combined with ahydrogel substrate to create an osteoinductive solution. A) Lyophilized and B)ground and combined with fibrin or C) alginate before (macroscopic) and after 6 weeksof ectopic implantation in mice (radiograph and safranin-O). Scale bar 3mm(Macroscopic) and 1 mm (Safranin-O). All conditions lead to the development ofmature bone tissues, including bone structures and a marrow compartment.Figure 5: Successful injectability of ground lyophilized extracellular matrixcombined with fibrinogen (fibrin) and thrombin. A). Example of gelation based onthe inverted vial method. B). Example of successfully mechanically self-sustainedhydrogels C).Ranges of successful injectability of ground lyophilized extracellularmatrix combined with fibrinogen (mg / mL) and thrombin (U / mL). In black, combination ofmixture unable to be successfully injected; in white, mixture demonstratingsuccessful injectability.Figure 6: The ground lyophilized extracellular matrix mixed with fibrin hydrogeland human mesenchymal stem cells promotes cartilage, fat, bone and bonemarrow tissue formation. A). Radiograph of a bone created from hMSCS after beinginjected subcutaneously with OssiGel after 6-weeks. B). Safranin-O staining of the Ossicle showing cortical bone surrounding cartilage, marrow, fat, and trabecular structures derived from human cells. C). Cartilage and D). marrow cavities within the bone.Figure 7: Healthy and human leukemia (AML) cells engraft in bones created withthe ground lyophilized extracellular matrix mixed with fibrin hydrogel. A). OssiGelallows the robust engraftment of healthy and leukemic human blood cells for thefundamental study of their behaviours and functions (e.g., proliferation, differentiation,heterogeneity). B) Healthy donor bone marrow cells mixed with Ossigel can form bonewith persisting bone and bone marrow elements post 8 weeks in vivo (trabecular bone,human mesenchymal cells, adipocytes and blood vessels). DAPI: nuclei, TdT : HumanHD-MSC and progeny, Perilipin: Adipocytes, Laminin: Vessels C). AML cells derivedfrom patients engrafted better in OssiGel-derived bones (squares) compared to thegold standard mice femurs (Mouse bone, circles). D). Explanted humanized bonesshowing the heterogeneity of AML samples. E). Different behavior observed in OssiGel-derived bone engrafted with Leukemia (top) when compared to non-engraftedP7250PC008bone (down). F) AML-derived MSC and leukemic cells can be visualized with aconfocal microscope 18 weeks post transplantation. DAPI: nuclei, TdT : Human AML-MSC and progeny, CD33 / CD45: Human leukemic cells. G-H) AML-derived MSC andleukemic cells can be quantified or sorted by flow cytometry / fluorescent activated cellsorting (FACS). Percentage of human leukemic white blood cells (huCD45+) in the ossicle (left) versus its corresponding mouse femur (right).Figure 8: Generation of in vivo ectopic bone via OssiGel mixed with prostatecancer patients derived mesenchymal stem cells (Pr-MSC). Prostate cancermesenchymal stem cells mixed with Ossigel forms bone with persisting bone and bonemarrow elements post 4 weeks in vivo (cortical bone with osteoblastic lining, humanmesenchymal stromal cells and their progeny, and blood vessels (A-B). DAPI: nuclei,TdT : Human Prostate-MSC and progeny, Vimentin: Human MSC, muCD31: Vessels.Microcomputed tomography (uCT) showed typical bone and bone marrow spaces(trabeculae) (C).Figure 9: Generation of ectopic in-vivo bone via OssiGel mixed with humaninduced pluripotent stem cells (iPSC) and induced pluripotent stem cell derivedmesenchymal stem cells (iPS-MSC). Both iPSC and iPS-MSC mixed with Ossigelform bone with persisting bone and bone marrow elements post 8 and 6 weeks in vivo (cortical bone with osteoblastic lining, human mesenchymal stromal cells and theirprogeny, and blood vessels. A) DAPI: nuclei, Vimentin: MSCs, muCD31: Vessels,huCD34: Hematopoietic and endothelial cells. B) DAPI: nuclei, TdT : Human HD-MSC and progeny, Endomucin: Vessels.Figure 10: Chondrogenic differentiation of human bone marrow-derivedmesenchymal stem cells (hBM-MSCs) and human mesenchymal stem cellsderived from induced pluripotent stem cells (hiPSC3-MSCs) using OssiGel. A)Histological analysis revealed enhanced Safranin-O positive red staining throughout the OssiGel group compared to the control counterpart, indicating an increase of GAG-rich cartilage matrix deposition. Figure 11: Evaluation of the dose-dependent effect of hBM-MSCs encapsulated inOssiGel on ectopic bone formation. OssiGel alone possess intrinsic osteoinductivepotential and enables efficient bone formation with minimal cell input. A) MacroscopicP7250PC009picture of explanted bones for each group after 6-weeks in vivo. B) uCT representative3D micrographs of explanted bones for each group after 6-weeks in vivo. C) BV / TV%quantification with uCT of explanted bones for each group after 6-weeks in vivo.Figure 12: Demonstration of chimeric bone formation derived from encapsulatedhBM-MSCs using OssiGel. A) Representative confocal images highlighting thechimeric origin of the humanized ossicles derived of the implantation of 1.000.000 hBM-MSCs with Ossigel. HuNu = human nuclei marker, specific to human cells, Osteocalcin= marker of mature osteoblasts and osteocytes, DAPI=nuclear DNA stain labeling all nucleated cells. Detailed description Definitions As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly states otherwise. The term “some embodiments” can include one, or more than one embodiment. The use of the word “a” or “an” when used throughout the text or in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”“Abundance” refers herein as the relative abundance used to determine the relativeatomic mass using mass spectrometry. “Cartilage” refers herein as a strong, flexible, non-vascular supporting connective tissuethat lines joints and protects the bones throughout the body. It may also refer to an in-vitro produced cartilage-like tissue. “Chondrogenesis” refers herein to the process through which cartilage tissue is formed and developed.P7250PC0010 “Chondrogenic factor” refers herein to a protein that promotes chondrogenesis, the formation of cartilage. “Differentiation” refers herein as the processes by which immature cells become mature cells with specific functions. “Endochondral ossification” refers herein as the process by which growing cartilage is systematically replaced by bone to form the growing skeleton. “Hydrogel” refers herein to a network of polymer chains in a gel in which the liquidcomponent is water. The person skilled in the art will also understand that a hydrogelcan also be referred to as a gel-like substrate. “Ossicle” refers herein to a small bone or bony structure. “Bone” and “ossicle” are used herein interchangeably.“OssiGel” refers herein to a composition comprising a ground extracellular matrix(ECM) produced by a human stem cell line, and a gel-like substrate (hydrogel) orprecursor thereof, as described in the present disclosure. “Osteogenesis” refers herein to the formation of bone. “Osteoinduction” refers herein to the process by which osteogenesis, bone formation, isinduced. In vivo, Osteoinduction may imply the recruitment of immature cells and thestimulation of these cells to develop into osteoblasts. “Vascularization” refers herein to the process of growing blood vessels into a tissue to improve oxygen and nutrient supply. CompositionsIn one aspect the invention relates to a ground extracellular matrix (ECM) produced bya human stem cell line.P7250PC0011In one aspect the human stem cell line has been differentiated to a chondrocyte, suchas a mature chondrocyte, and the ground extracellular matrix (ECM) is produced by thechondrocyte.In one aspect the invention relates to a ground extracellular matrix (ECM) produced bya human stem cell line with chondrogenic activity, wherein the human stem cell line is genetically modified to overexpress Bone Morphogenetic Protein-2 (BMP-2). “Extracellular matrix” (referred to as “ECM”) refers herein to a large network of proteins and other molecules that surround, support, and give structure to cells and tissues in the body. The extracellular matrix helps cells attach to, and communicate with, nearby cells, and plays an important role in cell growth, cell movement, and other cell functions.In a further aspect the invention relates to a composition comprising:a. the ECM as defined in the present disclosure, andb. a gel-like substrate or a precursor thereof.In a further aspect the invention relates to an injectable composition comprising:a. the ECM as defined in the present disclosure, andb. a hydrogel or a precursor thereof.In one embodiment, the composition induces formation of bone and / or bone marrow in-vivo.In one embodiment, the composition induces formation of bone and / or bone marrow in-vitro.“Bone” refers herein to a calcified tissue, or in a broader sense to bone as an organsystem, which comprises for example, but not limiting to, cells, such as adipocytes, osteocytes, chondrocytes, and precursors or further differentiation stages thereof, aswell as vascular and neural structures In other words, bone or bone as an organsystem can be regarded as skeletal and / or hematopoietic microenvironment.P7250PC0012 “Bone marrow” refers herein to the soft, spongy tissue that is in the medullary cavities (centers) of bones. Healthy bone marrow is an essential part of the body, as it contains stem cells that produce blood cells and immune cells.The composition of the invention (also referred to as “OssiGel”) offers a promising toolfor both in vitro and in vivo studies of bone and bone marrow formation. Thecomposition of the invention further offers a promising tool as a screening platform,decreasing time and costs for drug discovery and clinical trial time and cost.In one embodiment, the ground ECM has a particle size ranging from 1 to 1000 µm,such as ranging from 1 to 100 µm, such as ranging from 1 to 200 µm, ranging from 1 to 300 µm, ranging from 1 to 400 µm, ranging from 1 to 500 µm, ranging from 1 to 600 µm, ranging from 1 to 700 µm, ranging from 1 to 800 µm, ranging from 1 to 900 µm, such as ranging from 900 to 1000 µm, such as ranging from 800 to 1000 µm, such as ranging from 700 to 1000 µm, such as ranging from 600 to 1000 µm, such as ranging from 500 to 1000 µm, such as ranging from 400 to 1000 µm, such as ranging from 300 to 1000 µm, such as ranging from 200 to 1000 µm, such as ranging from 100 to 1000 µm.In one embodiment, the human stem cell line is a human mesenchymal stem cell line."Mesenchymal stem cells” (referred to as "MSCs") are referred to herein as being multipotent stromal cells that can differentiate into a variety of cell types, including but not limited to: osteoblasts (bone cells), chondrocytes (cartilage cells), and adipocytes (fat cells).In one embodiment, the human mesenchymal stem cell line is a stem cell lineoverexpressing Bone Morphogenetic Protein-2 (BMP-2).In one embodiment, the stem cell line overexpressing BMP-2 is the MesenchymalSword of Damocles – BMP2 (MSOD-B) cell line.The MSOD-B line is genetically modified to overexpress Bone Morphogenetic Protein-2(BMP-2)P7250PC0013The MSOD-B line is lentivirally transduced for constitutive expression of the humanBMP-2 transgene.In one embodiment, the precursor comprises non-dissolved components of the gel-likesubstrate. The non-dissolved components of the gel-like substrate can for example be a dry powder of the ingredients, such as fibrinogen, thrombin, alginate and / or polyethylene glycols.The person skilled in the art knows how to dissolve the components in for examplebuffer or water to create the gel-like substrate. In one embodiment, the components are selected from the group consisting of fibrinogen, thrombin, alginate and / or polyethylene glycols.In one embodiment, the gel-like substrate or precursor thereof is synthetic and / ornaturally occurring.Naturally occurring hydrogels are three-dimensional, water-swollen polymer networksderived from natural materials like proteins and polysaccharides. They are formedwhen these natural polymers crosslink and interact with water. In one embodiment, the naturally occurring gel-like substrate or precursor thereof comprises fibrinogen and / or thrombin.In one embodiment, the gel-like substrate or precursor thereof comprises fibrinogen.In one embodiment, thrombin is added to the gel-like substrate or precursor thereofcomprising fibrinogen to induce cross-linking and / or polymerization.In one embodiment, thrombin is added to the gel-like substrate or precursor thereofcomprising fibrinogen prior to in vivo injection of the composition, such as 10 minutes, such as 9 minutes, such as 8 minutes, such as 7 minutes, such as 6 minutes, such as 5 minutes, such as 4 minutes, such as 3 minutes, such as 2 minutes, such as 1 minute prior to in vivo injection.P7250PC0014In one embodiment, thrombin is added to the gel-like substrate or precursor thereofcomprising fibrinogen 5 minutes prior to in vivo injection of the composition.In one embodiment, the naturally occurring gel-like substrate or precursor thereofcomprises or consists of alginate.In one embodiment, the synthetic gel-like substrate or precursor thereof comprises orconsists of polyethylene glycols.In one embodiment, the synthetic gel-like substrate or precursor thereof comprises 8 to40 % polyethylene glycols. In one embodiment, the gel-like substrate or precursor thereof comprises at least 5 mg / ml fibrinogen, such a 6 mg / ml, such as 7 mg / ml, such as 8 mg / ml, such as 9 mg / ml, such as 10 mg / ml, such as 11 mg / ml, such as 12 mg / ml, such as 13 mg / ml, such as 14 mg / ml, such as 15 mg / ml, such as 16 mg / ml, such as 17 mg / ml, such as 18 mg / ml, such as 19 mg / ml, such as 20 mg / ml.In one embodiment, the gel-like substrate or precursor thereof comprises 7 to 15 mg / mlfibrinogen.In one embodiment, the gel-like substrate or precursor thereof comprises at least 0.5U / ml thrombin, such as 0.6 U / ml, such as 0.7 U / ml, such as 0.8 U / ml, such as 0.9 U / ml, such as 1 U / ml, such as 1.1 U / ml, such as 1.2 U / ml, such as 1.3 U / ml, such as 1.4 U / ml, such as 1.5 U / ml, such as 1.6, U / ml, such as 1.7, U / ml, such as 1.8, U / ml, such as 1.9, U / ml, such as 2 U / ml, such as 2.1 U / ml, such as 2.2 U / ml, such as 2.3 U / ml, such as 2.4 U / ml, such as 2.5 U / ml, such as 2.6, U / ml, such as 2.7, U / ml, such as 2.8, U / ml, such as 2.9, U / ml, such as 3 U / ml.In one embodiment, the gel-like substrate or precursor thereof comprises 1 to 1.5 U / mlthrombin.In one embodiment, the composition comprises at least 0.1 mg ground ECM, such as0.2 mg, such as 0.3 mg, such as 0.4 mg, such as 0.5 mg, such as 0.5 mg, such as 0.6 mg, such as 0.7 mg, such as 0.8 mg, such as 0.9 mg, such as 1 mg, such as 2 mg,P7250PC0015 such as 3 mg, such as 4 mg, such as 5 mg , such as 6 mg, such as 7 mg, such as 8 mg, such as 9 mg, such as 10 mg.In one embodiment, the composition comprises 1 mg ground ECM.In one embodiment, the composition comprises or consists of 1 mg ground ECM, 7.5-wt % human fibrin and 0.9 U / mL human thrombin.In one embodiment, the composition further comprises cells.In one embodiment, the cells are human cells.In one embodiment, the human cells are human stem cells.In one embodiment, the human cells are human stem cells.In one embodiment, the human stem cells are human bone marrow-derivedmesenchymal stem cells (hBM-MSCs), and / or human pluripotent stem cells.In one embodiment, the human pluripotent stem cells are induced pluripotent stemcells (iPCS).In one embodiment, the human stem cells further comprised in the composition arehuman mesenchymal stem cells.In one embodiment, the human cells are human cancer cells.In one embodiment, the cancer cells are leukemia cells.In one embodiment, the cancer cells are solid cancer cells.In one embodiment, the leukemia cells are selected from the group consisting of acutemyeloid leukemia (AML) cells, acute lymphocytic leukemia (ALL) cells, chronic lymphocytic leukemia (CLL) cells, and chronic myeloid leukemia (CML) cells.In one embodiment, the solid cancer cells are selected from the group consisting ofbone cancer cells, prostate cancer cells, breast cancer cells, and neuroblastoma cells.P7250PC0016In one embodiment, the leukemia cells are AML cells.In one embodiment, the AML cells are mononucleated cells, such as CD45+ / CD3-AML cells.In one embodiment, the AML cells are isolated from the blood or bone marrow of AMLpatients.In one embodiment, the composition further comprises at least 2 µg / mlglycosaminoglycans, such as 3 µg / ml, such as 4 µg / ml, such as 5 µg / ml, such as 6 µg / ml, such as 7 µg / ml, such as 8 µg / ml, such as 9 µg / ml, such as 10 µg / ml, such as 11 µg / ml, such as 12 µg / ml, such as 13 µg / ml, such as 14 µg / ml, such as 15 µg / ml, such as 16 µg / ml, such as 17 µg / ml, such as 18 µg / ml, such as 19 µg / ml, such as 20 µg / ml.In one embodiment, the composition further comprises at least 10 µg / ml BoneMorphogenetic Protein-2 (BMP-2), such as 15 µg / ml, such as 20 µg / ml, such as 25 µg / ml, such as 30 µg / ml, such as 35 µg / ml, such as 40 µg / ml such as 45 µg / ml, such as 50 µg / ml, such as 55 µg / ml, such as 60 µg / ml.In one embodiment, the ground ECM and the gel-like substrate or precursor thereof aremixed with each other.The ground ECM and the gel-like substrate or precursor thereof can be mixed in asyringe, for in-vivo injection, or in a test tube, for storage and / or in vitro studies. The person skilled in the art will understand that polymerization happens whenthrombin is added to the mixture of cells, fibrinogen and ground ECM. This processinvolves the cleavage of fibrinopeptides A and B from the fibrinogen molecule, converting it into fibrin monomers. These monomers spontaneously polymerize to form a fibrin network through non-covalent interactions, creating an initial fibrin gel. Theconcentration of fibrinogen and thrombin is optimized to ensure a controlled rate ofpolymerization. Typically, lower thrombin concentrations slow the polymerizationprocess, allowing sufficient time for injection. The polymerization reaction isP7250PC0017 temperature-dependent. Lower temperatures can slow down the reaction, extending the workable time before the solution fully polymerizes.In one embodiment, the composition further comprises cell culture media.In one embodiment, the cell culture media is Dulbecco’s modified eagle’s medium(DMEM). KitIn another aspect the invention relates to a kit comprising the composition as defined inthe present disclosure, phosphate-buffered saline, cell culture media, and instructions for use.In one embodiment, the kit further comprises thrombin.In one embodiment, the kit further comprises cells.A precursor of the gel-like substrate, as defined in the present disclosure, can be forexample a dry powder of the ingredients. The dry powder and ECM can be part of the,as defined in the present disclosure, kit. The dry powder can be dissolved for examplewith buffer, water and / or other ingredients of the kit.MethodsIn another aspect the invention relates to a method of manufacturing the compositionas defined in the present disclosure, the method comprising the following steps: a. culturing a stem cell line in vitro;b. exposing the stem cell line to differentiation factors, therebydifferentiating said stem cell line into mesenchymal lineage cell types; c. culturing the mesenchymal lineage cell types, thereby producing anextracellular matrix (ECM); d. lyophilizing the cartilage cells comprised in the ECM of step c, therebyobtaining said ECM; e. grinding the ECM of step d, thereby obtaining a ground ECM;and / or f. mixing the ground ECM with a gel-like substrate or precursor thereof.P7250PC0018 “Lyophilization” refers herein as freeze-drying, a form of dehydration, where a material first is frozen, then dried under pressure to remove its water content.“Grinding” refers herein as the reduction of a substance or material to small particles orpowder by for example crushing, milling or pressing. The person skilled in the art willunderstand that any method that is suitable for producing a powder can be used to prepare the ground ECM of the invention. In one embodiment, the stem cell line is cultured on 2D culture plates. In one embodiment, the stem cell line is cultured on 3D porous scaffolds. In one embodiment, the 3D porous scaffolds are collagen scaffolds, such as sponges. In one embodiment, the collagen is collagen type I.In one embodiment, the human stem cell line is a human mesenchymal stem cell line.In one embodiment, the human mesenchymal stem cell line is a stem cell lineoverexpressing Bone Morphogenetic Protein-2 (BMP-2).In one embodiment, the stem cell line overexpressing BMP-2 is the MesenchymalSword of Damocles – BMP2 (MSOD-B) cell line.In one embodiment, the differentiation factors are chondrogenic factors. In one embodiment, the chondrogenic factors are selected from the group consistingof, transferrin, selenium, ascorbic acid, dexamethasone, TGF-β3, TGF-β1, BMP-2,BMP-7, and BMP-4.In one embodiment, the ascorbic acid concentration is at least 0.01 mM, such as 0.05mM, such as 0.1 mM, such as 0.2 mM, such as 0.5 mM.In one embodiment, the dexamethasone concentration is at least 0.01 µM, such as0.05 µM, such as 0.1 µM, such as 0.5 µM, such as 1 µM, such as 1.5 µM, such as 2 µM.P7250PC0019In one embodiment, the TGF-β3 concentration is at least 1 ng / ml, such as 2 ng / ml,such as 5 ng / ml, such as 10 ng / ml, such as 15 ng / ml, such as 20 ng / ml. In one embodiment, the stem cell line is exposed to differentiation factors for at least 3days, such as at least 4 days, such as at least 5 days, such as at least 6 days, such asat least 7 days, such as at least 8 days, such as at least 9 days, such as at least 10days, such as at least 11 days, such as at least 12 days, such as at least 13 days, suchas at least 14 days, such as at least 15 days, such as at least 16 days, such as at least17 days, such as at least 18 days, such as at least 19 days, such as at least 20 days,such as at least 21 days, such as at least 22 days, such as at least 23 days, such as atleast 24 days, such as at least 25 days, such as at least 26 days, such as at least 27days, such as at least 28 days, such as at least 29 days, such as at least 30 days, suchas at least 31 days, such as at least 32 days, such as at least 33 days, such as at least34 days, such as at least 35 days.In one embodiment, the stem cell line is exposed to differentiation factors for at least 1 week, such as at least 2 weeks, such as at least 3 weeks, such as at least 4 weeks, such as at least 5 weeks. In one embodiment, the cells are further exposed to penicillin-streptomycin-glutamine, sodium pyruvate, and insulin. In one embodiment, the mesenchymal lineage cell types are selected from the group consisting of bone cells, fat cells, cartilage cells, stromal cells and / or muscle cells.Cartilage cells may also be referred to as chondrocytes and / or chondroblasts.Bone cells may also be referred to as osteoblasts and / or osteocytes.Fat cells may also be referred to as adipocytes.Muscle cells may also be referred to as myocytes and / or myoblasts.In one embodiment, the cells are rinsed with PBS before lyophilization. In one embodiment, the cells are snap frozen for 5 minutes in liquid nitrogen before lyophilization.P7250PC0020 In one embodiment, the cartilage tissue is lyophilized overnight. In one embodiment, the cartilage tissue is lyophilized at -80°C.In one embodiment, the cartilage tissue is lyophilized with a pressure of 0.05 mbar.In one embodiment, the ECM is ground with the CryoMill.In one embodiment, the ECM is ground in a grinding jar.In one embodiment, the grinding jar has a volume of 10 ml.In one embodiment, the grinding jar is of stainless steel.In one embodiment, the grinding jar comprises two 10 mm grinding balls.In one embodiment, the ECM is ground for 4x 2 minutes.In one embodiment, the ECM is ground at 25 hz.In one embodiment, the ground ECM has a particle size ranging from 1 to 1000 µm, such as ranging from 1 to 100 µm, such as ranging from 1 to 200 µm, ranging from 1 to 300 µm, ranging from 1 to 400 µm, ranging from 1 to 500 µm, ranging from 1 to 600 µm, ranging from 1 to 700 µm, ranging from 1 to 800 µm, ranging from 1 to 900 µm, such as ranging from 900 to 1000 µm, such as ranging from 800 to 1000 µm, such as ranging from 700 to 1000 µm, such as ranging from 600 to 1000 µm, such as ranging from 500 to 1000 µm, such as ranging from 400 to 1000 µm, such as ranging from 300 to 1000 µm, such as ranging from 200 to 1000 µm, such as ranging from 100 to 1000 µm.In one embodiment, the gel-like substrate or precursor thereof is synthetic and / ornaturally occurring.In one embodiment, the naturally occurring gel-like substrate or precursor thereofcomprises fibrinogen and / or thrombin.In one embodiment, the naturally occurring gel-like substrate or precursor thereofcomprises alginate.P7250PC0021In one embodiment, the synthetic gel-like substrate or precursor thereof comprises 8 to40 % polyethylene glycols.In one embodiment, the gel-like substrate or precursor thereof comprises at least 5mg / ml fibrinogen, such a 6 mg / ml, such as 7 mg / ml, such as 8 mg / ml, such as 9 mg / ml,such as 10 mg / ml, such as 11 mg / ml, such as 12 mg / ml, such as 13 mg / ml, such as 14mg / ml, such as 15 mg / ml, such as 16 mg / ml, such as 17 mg / ml, such as 18 mg / ml,such as 19 mg / ml, such as 20 mg / ml.In one embodiment, the gel-like substrate or precursor thereof comprises 7 to 15 mg / mlfibrinogen.In one embodiment, the gel-like substrate or precursor thereof comprises at least 0.5U / ml thrombin, such as 0.6 U / ml, such as 0.7 U / ml, such as 0.8 U / ml, such as 0.9 U / ml, such as 1 U / ml, such as 1.1 U / ml, such as 1.2 U / ml, such as 1.3 U / ml, such as 1.4 U / ml, such as 1.5 U / ml, such as 1.6, U / ml, such as 1.7, U / ml, such as 1.8, U / ml, such as 1.9, U / ml, such as 2 U / ml, such as 2.1 U / ml, such as 2.2 U / ml, such as 2.3 U / ml, such as 2.4 U / ml, such as 2.5 U / ml, such as 2.6, U / ml, such as 2.7, U / ml, such as 2.8, U / ml, such as 2.9, U / ml, such as 3 U / ml.In one embodiment, the gel-like substrate or precursor thereof comprises 1 to 1.5 U / mlthrombin. In one embodiment, the composition comprises at least 0.1 mg ground ECM, such as 0.2 mg, such as 0.3 mg, such as 0.4 mg, such as 0.5 mg, such as 0.5 mg, such as 0.6 mg, such as 0.7 mg, such as 0.8 mg, such as 0.9 mg, such as 1 mg, such as 2 mg, such as 3 mg, such as 4 mg, such as 5 mg , such as 6 mg, such as 7 mg, such as 8 mg, such as 9 mg, such as 10 mg. In one embodiment, the composition comprises 1 mg ground ECM. In one embodiment, the composition further comprises at least 2 µg / mlglycosaminoglycans, such as 3 µg / ml, such as 4 µg / ml, such as 5 µg / ml, such as 6µg / ml, such as 7 µg / ml, such as 8 µg / ml, such as 9 µg / ml, such as 10 µg / ml, such as11 µg / ml, such as 12 µg / ml, such as 13 µg / ml, such as 14 µg / ml, such as 15 µg / ml,such as 16 µg / ml, such as 17 µg / ml, such as 18 µg / ml, such as 19 µg / ml, such as 20µg / ml.P7250PC0022In one embodiment, the composition further comprises at least 10 µg / ml BoneMorphogenetic Protein-2 (BMP-2), such as 15 µg / ml, such as 20 µg / ml, such as 25µg / ml, such as 30 µg / ml, such as 35 µg / ml, such as 40 µg / ml such as 45 µg / ml, suchas 50 µg / ml, such as 55 µg / ml, such as 60 µg / ml.In one embodiment, the methods as defined in the present disclosure further comprisescell culture media.In one embodiment, the cell culture media is Dulbecco’s modified eagle’s medium(DMEM).In another aspect the invention relates to an in vivo method for bone formation, themethod comprising the following steps: a. administering the composition as defined in the present disclosure to asubject; and b. maintaining for at least 1 week, such as 2 weeks, such as 3 weeks.In another aspect the invention relates to an in vivo method for bone and bone marrowtissue formation, the method comprising the following steps:a. administering the composition as defined in the present disclosure to asubject; and b. maintaining for at least 2 weeks, such as 3 weeks, 4 weeks, such as 5weeks, such as 6 weeks, such as 7 weeks, such as 8 weeks.In one embodiment, the subject is a mammal.In one embodiment, the mammal is a mouse, rat, rabbit, pig, dog, guinea pig ormonkey.In one embodiment, the administration in vivo is a subcutaneous injection.The method, wherein the administration in vivo is an implantation.P7250PC0023In one embodiment, at least 1 mm3, such as 2 mm3, such as 3 mm3, such as 4 mm3,such as 5 mm3, such as 6 mm3, such as 7 mm3, such as 8 mm3, such as 9 mm3, such as 10 mm3, such as 11 mm3, such as 12 mm3, such as 13 mm3, such as 14 mm3, such as 15 mm3, such as 16 mm3, such as 17 mm3, such as 18 mm3, such as 19 mm3, such as 20 mm3bone tissue is formed, at least 1 week, such as 2 weeks, such as 3 weeks, such as 4 weeks, such as 5 weeks, such as 6 weeks post-in vivo administration.In one embodiment, the amount of bone tissue formation is assessed bymicrotomography analysis.In one embodiment, the methods as defined in the present disclosure furthercomprising administering cells. In one embodiment, the administered cells are human mesenchymal stem cells(hMSCs), genetically engineered cell lines, human hematopoietic stem / progenitor cells(HSPCs), human cancer stem cells, and / or human immune cells.In one embodiment, the human mesenchymal stem cells (hMSCs) are bone marrow-derived MSCs, adipose-derived MSCs (ASCs), and / or MSCs derived from inducedpluripotent stem cells (hiPSC-MSCs).In one embodiment, the human cancer stem cells are multiple myeloma,osteosarcoma, metastatic breast cancer, metastatic prostate cancer, or leukemia cells.In another aspect the invention relates to an in vitro method for bone and / or bonemarrow tissue formation, the method comprising the following steps: a. placing the composition as defined in the present disclosure in culturemedia; b. administering cells;c. incubating for at least 1 week, such as 2 weeks, such as 3 weeks, suchas 4 weeks, such as 5 weeks, such as 6 weeks, such as 7 weeks, such as 8 weeks. The cells can be mixed directly with the compound of the invention, administered in- vivo after bone and bone marrow formation or added after in vitro polymerization.In one embodiment, the cells are human cells.P7250PC0024 The compound of the invention offers a promising tool for in vitro studies. Leveraging the hydrogel’s extracellular matrix (ECM) properties and growth factors, cell proliferation, differentiation and essential processes of endochondral ossification canalso be studied in vitro. The in vitro use of the compound of the invention is anticipatedto support robust proliferation of hMSCs and their differentiation into chondrocytes. The cartilaginous matrix composition provides a conducive ECM-rich environment that promotes cellular adhesion, growth, and differentiation. The potential of this technology will enable detailed analysis of the endochondral ossification process, including the critical stages of chondrogenic differentiation and hypertrophy. An example could be the insertion of specific cell mutations using CRISPR / Cas9 or other gene-editing technologies to study their impact on chondrogenic differentiation and hypertrophy. These processes could provide insights into developmental bone disorders and potential therapeutic targets.In one embodiment, the human cells are human stem cells.In one embodiment, the human cells are human mesenchymal stem cells.In one embodiment, the human cells are human cancer cells.In one embodiment, the cancer cells are leukemia cells.In one embodiment, the cancer cells are solid cancer cells.In one embodiment, the leukemia cells are selected from the group consisting of acutemyeloid leukemia (AML) cells, acute lymphocytic leukemia (ALL) cells, chronic lymphocytic leukemia (CLL) cells, and chronic myeloid leukemia (CML) cells.In one embodiment, the solid cancer cells are selected from the group consisting ofbone cancer cells, prostate cancer cells, breast cancer cells, and neuroblastoma cells.In one embodiment, the leukemia cells are AML cells.The AML cells can be mononucleated cells, such as CD45+ / CD3- AML cells, isolatedfrom the blood or bone marrow of AML patients.P7250PC0025In one embodiment, the formation of bone and bone marrow is identified usingradiographic imaging.In one embodiment, the formation of bone and bone marrow is identified using micro-computed tomography (µCT).In one embodiment, the formation of bone and bone marrow is identified usingSafranin-O / Fast Green staining.In one embodiment, the formation of bone and bone marrow is identified usingMasson's Trichrome staining.In one embodiment, human cells are present in the formed bone marrowmicroenvironment, at least 1 week, such as at least 2 weeks, such as at least 3 weeks, such as at least 4 weeks, such as at least 5 weeks, such as at least 6 weeks post-in vivo implantation. In another aspect the invention relates to a method for testing a candidate pharmaceutical product, the method comprising the following steps: a. establishing bone and bone marrow;b. administering pathological cells to the bone and bone marrow of step a,thereby establishing a model pathological bone and bone marrow;c. assessing the viability and / or cell number of the pathological cells;d. administering a candidate pharmaceutical product;e. assessing the viability and / or cell number of the pathological cells;and f. classifying the candidate pharmaceutical product as effective againstpathological cells if the viability and / or cell number is decreased in step e compared to step c. In one embodiment, the candidate pharmaceutical product is a pharmaceutical product against engrafted leukemia blood cells and / or engrafted human solid cancer cells.In one embodiment, the pathological cells are patient-derived pathological cells orgenetically engineered pathological cells.P7250PC0026In one embodiment, the patient-derived pathological cells are primary cancer cells andtheir premalignant state cells, hematopoietic cells from patients with genetic disorders, marrow failure, or clonal hematopoiesis, tumor-initiating cells or cancer stem cells; and / or inflammatory or immune cells from autoimmune bone pathologies. In one embodiment, the genetically engineered pathological cells are mesenchymal cells, hematopoietic cells, epithelial or carcinoma-derived cells, pluripotent stem cells differentiated into bone, marrow, or cancer-relevant lineages, immortalized cell lines or patient-derived xenograft (PDX) derivatives. In one embodiment, the mesenchymal cells are MSCs and / or stromal cells. In one embodiment, the hematopoietic cells are HSCs, progenitors, and / or lineage- committed immune cells. In one embodiment, the epithelial or carcinoma-derived cells are breast, prostate, or lung cancer cells with bone metastatic potential.In one embodiment, the pluripotent stem cells differentiated into bone, marrow, orcancer-relevant lineages are hiPSCs.In one embodiment, the leukemia cells are selected from the group consisting of acutemyeloid leukemia (AML) cells, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML).In one embodiment, the solid cancer cells are selected from the group consisting ofbone cancer cells, prostate cancer cells, breast cancer cells, and neuroblastoma cells.In one embodiment, the leukemia cells are AML cells.The AML cells can be mononucleated cells, such as CD45+ / CD3- AML cells, isolatedfrom the blood or bone marrow of AML patients. In another aspect the invention relates to a method for identifying treatmentresponders, the method comprising the following steps:a. establishing bone and bone marrow;P7250PC0027 b. isolating cells from a patient;c. administering the isolated cells from step b) to the bone andbone marrow of step a), thereby establishing a model of the bone and bone marrow of said patient; d. assessing the viability and / or cell number of the administeredcells in the bone marrow; e. administering a pharmaceutical product;f. assessing the viability and / or cell number of the administeredcells; and g. classifying the patient as a responder to the pharmaceuticalproduct if the viability and / or cell number is decreased in step f) compared to step d.In another aspect the invention relates to a method for identifying treatmentresponders, the method comprising the following steps:a. obtaining cells from a patient, wherein the cells are primarycancer cells and their premalignant state cells, hematopoieticcells from patients with genetic disorders, marrow failure, or clonal hematopoiesis, tumor-initiating cells or cancer stem cells, and / or inflammatory or immune cells from autoimmune bone pathologies; b. mixing the cells of step a. with the composition as defined in thepresent disclosure; c. administering the mixture of step b. to a subject;d. maintaining the mixture in the subject for at least 2 weeks, suchas 3 weeks, 4 weeks, such as 5 weeks, such as 6 weeks, such as 7 weeks, such as 8 weeks, thereby establishing a model ofthe bone and bone marrow of said patient; e. assessing the viability and / or cell number of the administeredcells in the bone marrow; f. administering a pharmaceutical product;g. assessing the viability and / or cell number of the administeredcells; andP7250PC0028 classifying the patient as a responder to the pharmaceutical product if the viability and / or cell number is decreased in step g compared to step e. In one embodiment, the candidate pharmaceutical product is a drug. In one embodiment, the candidate pharmaceutical product is an agent used in cell therapy or gene therapy. In one embodiment, the viability and / or cell number of the pathological cells isassessed using hemocytometry, flow cytometry, confocal microscopy, histopathologicalanalysis, such as immunohistochemistry, and / or transcriptomic profiling, such as RNA sequencing.In one embodiment, the viability and / or cell number of the pathological cells isassessed using hemocytometry.In one embodiment, the viability and / or cell number of the pathological cells isassessed using flow cytometry.In one embodiment, the viability and / or cell number of the pathological cells isassessed using FACS.In one embodiment, the viability and / or cell number of the pathological cells isassessed using confocal microscopy.In one embodiment, the viability and / or cell number of the pathological cells isassessed using histopathological analysis, such as immunohistochemistry.In one embodiment, the viability and / or cell number of the pathological cells isassessed using the markers CD34, CD117, CD33, CD45RA, and / or CD123.In one embodiment, the viability and / or cell number of the pathological cells isassessed using transcriptomic profiling, such as RNA sequencing.P7250PC0029In one embodiment, presence of ≥1% pathological cells is indicative of a pathologicalbone and bone marrow. In one embodiment, the cells are cancer cells. In one embodiment, the cancer cells are multiple myeloma, leukemia, metastatic solid tumors. Medical useIn one embodiment, the composition as defined in the present disclosure, is for use inmedicine. In one embodiment, the composition as defined in the present disclosure, is for use in a method of treating cancer. In one embodiment, the cancer is selected from the group consisting of leukemia, andsolid cancer. In one embodiment the cancer is selected from hematological cancersand their precursors. The leukemia and / or the hematological cancer cells and their premalignant conditions cells are selected from the group consisting of acute myeloid leukemia (AML) cells, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), and Chronic myeloid leukemia (CML), multiple myeloma (MM) cells, myelodysplastic syndromes(MDS) cells, and myeloproliferative neoplasm (MPN) cells such as AML cells.The solid cancer cells can be for example, but not limited to, bone cancer cells,prostate cancer cells, breast cancer cells, and neuroblastoma cells. “Treating,” or “Treatment,” refers herein to any administration or application of a therapeutic for the disclosed diseases, disorders and conditions in subject, and includes inhibiting the progression of the disease, slowing the disease or its progression, arresting its development, partially or fully relieving the disease, or partially or fully relieving one or more symptoms of a disease. ProductIn another aspect the invention relates to a product obtained by the methods asdisclosed herein.P7250PC0030 Examples Example 1: Development and Characterization of OssiGel: A Custom Hydrogelfor Ectopic Humanized Bone and Bone Marrow Formation in Nude Mice forCancer Research Aim: Provide a custom hydrogel (OssiGel) that when injected ectopically into the back of nude mice, can establish bone tissue with a developed human-derived bone marrow environment, in a xenograft setup to study bone-developing cancers, such as leukemia or solid cancers metastasizing in bones. Material and Methods: MSOD-B cells, a stable human mesenchymal cell line capable of cartilage formation through constitutive BMP-2 expression, were seeded at a density of 2 million cells in 35 µL on type I collagen sponges previously punched to a diameter of 6 mm (Avitene Ultrafoam, BD). After a 1h incubation time at 37 °C, constructs were primed toward chondrogenic differentiation for 3 weeks to achieve cartilage tissue formation. Chondrogenic medium consisted of DMEM supplemented with penicillin streptomycin- glutamine (Gibco), HEPES (Gibco), sodium pyruvate (Gibco), ITS-A (insulin, transferrin, selenium) (Gibco), human serum albumin 0.12% (CSL Behring), 0.1 × 10−3Μ ascorbic acid (A5960, Sigma), 10−7Μ dexamethasone (D4902, Sigma) and 10 ng mL−1TGF-β3 (Novartis). Following the 3-weeks in vitro differentiation, samples were rinsed with PBS, snap frozen for 5 minutes in liquid nitrogen and lyophilized overnight at -80°C with a pressure of 0.05mbar. Lyophilized cartilages were then ground with the CryoMill in a 10 ml grinding jar of stainless steel with two 10 mm grinding balls. In order to ensure that the samples were pre-embrittled before grinding a pre-cooling time of 12 minutes was performed. Grinding was then performed for 4x 2 minutes at 25 hz and an intermediate cooling time of 30 seconds at 5Hz.The ground extracellular matrix (ECM) was mixed with fibrinogen, cell culture mediaand human cells, in a suitable container (i.e. syringe). Thrombin was then added to themixture to promote fibrinogen cross-linking into fibrin hydrogel (Figure 1A). The addition of thrombin into the mixture promotes the cleavage of fibrinopeptides A and B from the fibrinogen molecule. This exposes the polymerization sites on the fibrinogen molecule, which allows for the formation of fibrin monomers. The fibrin monomers then undergoP7250PC0031 polymerization and lateral aggregation, which results in the formation of a fibrin network. The network is stabilized through the formation of covalent bonds between the fibrin monomers, which is facilitated by the action of thrombin. The resulting fibrinnetwork is a strong and stable structure that encapsulate the ground ECM and thehuman cells. The present invention describes a hydrogel composed of 1mg ground ECM, 7.5-wt % human fibrin, 0.9U / mL human thrombin and human cells at a desired number which polymerizes within 2 minutes at 37οC upon addition of thrombin into the mixture.The method comprises injecting the mixture described above (200ul per dosage) tomice, within 5 minutes of adding thrombin to the mixture. One advantage of the present invention is that the composition can be injected into a desired site, which allows for targeted delivery and minimally invasive procedures. After a period of 2 weeks, bone formation was observed at the site of injection, indicating the osteogenic potential of the OssiGel. After a period of 4 weeks, the establishment of a mature bone with bone marrow was observed. Importantly, the embedded human cells differentiated in multi-lineages establishing a human micro-environment. This indicated the ability of the hydrogel to support the growth anddifferentiation of injected human cells towards human tissue formation (Figure 1B).The present invention can also be used for in-vitro bone formation. Bone formation isstudied in-vitro by incubating the mixture described above in cell culture media withhuman mesenchymal stem cells for 2 to 6 weeks. The cartilaginous matrix composition provides a conductive ECM-rich environment that promotes cellular adhesion, growth,and differentiation, and can be used to study cell proliferation, differentiation andessential processes of endochondral ossification in vitro (Figure 1C). Results:OssiGel promotes formation of bone and a bone marrow microenvironment in vivo andin vitro. OssiGel promotes the differentiation of human cells towards the establishmentof a bone marrow niche.P7250PC0032 Conclusion: A human-derived marrow environment in a xenograft setup to study bone-developing cancers, such as leukemia or solid cancers metastasizing in bones, was established.Example 2: Analysis of particle size distribution in lyophilized and ground humanextracellular matrix for hydrogel applicationsAim:Investigate the size distribution of the lyophilized and ground extracellular matrix(ECM). Material and Methods: A human mesenchymal stem cell line, MSOD-B, was seeded on 3D porous collagen scaffolds as described above. In short, the cells on the scaffolds were exposed to chondrogenic factors, for 3 weeks. After 3 weeks of in vitro differentiation, the resultinghuman cartilage tissue was lyophilized. The lyophilized ECM was imaged using a high-resolution mobile digital camera under consistent lighting conditions to ensureuniformity in the analysis. The lyophilized ECM was further ground in order to obtain afine ECM. The lyophilized, ground ECM was imaged as described above, and usingscanning electron microscopy (SEM).Results: The particles of the lyophilized cartilage tissue were spherical and around 3 mm indiameter (Figure 2A). The lyophilized, ground ECM was a fine powder (Figure 2B)consisting of small particles ranging from 1 to 1000 µm (Figure 2C). The fine powderconsists of small particles and big particles, ranging from 6 to 200 μm (Figure 2D). Thedata represent Mean ^ SD on a logarithmic scale. Small particles have a mean size of6.7 ^ 14.5 micrometers and big particles have a mean size of 272 ^ 226 micrometers.Conclusion: The particles of the lyophilized cartilage are large, whereas the lyophilized, groundECM is a fine powder consisting of smaller particles ranging from 1 to 1000 µm,resulting in injectability.P7250PC0033Example 3: Assessment of glycosaminoglycan (GAG) retention during andfollowing the grinding of lyophilized human cartilage tissueAim:Determine the retention of GAGs during and after the grinding process of thelyophilized cartilage tissue. Material and Methods: A human mesenchymal stem cell line, MSOD-B, was seeded on 3D porous collagen scaffolds as described above. In short, the cells on the scaffolds were exposed to chondrogenic factors, for 3 weeks. After 3 weeks of in vitro differentiation, the resulting human cartilage tissue was lyophilized. The lyophilized cartilage tissue was furtherground in order to obtain a fine extracellular matrix (ECM) powder. The amount(concentration) of GAGs in the lyophilized ECM (µg GAGs / mg dry weight ECM) wasmeasured before and after grinding, using Blyscan kit (Biocolor Ltd., UK). Briefly, samples were digested overnight at 56ºC in 0.5mL Proteinase K solution (Sigma Aldrich, P2308), and 20^L of each sample were assayed following the manufacturer’s protocol. Results:The amount (concentration) of GAGs in the lyophilized cartilage was maintained duringthe grinding process (Figure 3). Conclusion:The amount (concentration) of GAGs in the lyophilized cartilage was maintained duringthe grinding process, allowing successful bone and bone marrow formation. Example 4: Retention of osteoinductive properties after extracellular matrix lyophilization, grinding, and mixing with a hydrogel substrate. Aim:Investigate the lyophilized and ground extracellular matrix (ECM) before and aftermixing with fibrin or alginate.P7250PC0034 Material and Methods: A human mesenchymal stem cell line, MSOD-B, was seeded on 3D porous collagen scaffolds as described above. In short, the cells on the scaffolds were exposed to chondrogenic factors, for 3 weeks. After 3 weeks of in vitro differentiation, the resultinghuman cartilage tissue was lyophilized. The lyophilized ECM was imaged using a high-resolution mobile digital camera under consistent lighting conditions to ensure uniformity in the analysis; on a Zeiss Axiophot microscope; and with the U-CT system (MILabs, The Netherlands) using a tungsten x-ray source at 50 kV and 0.21 mA. Thelyophilized ECM was further ground in liquid nitrogen with a pestle and mortar. Equalamount of powder was divided in 1,5 mL Eppendorfs and centrifuged at high speed for 1min and stored in ice until proceeding with gel encapsulation. For fibrin encapsulation, 35uL of 40mg / ml Fibrinogen (Baxter, Switzerland) were added into each Eppendorf (n=6). Reticulation occurs after the addition of 35uL of 12IU / ml of thrombin (Baxter, Switzerland). Jellification takes 5min at 37°C. Once reticulated, gels are removed and kept in PBS 1X 7,2 (Gibco Invitrogen, USA) till implantation. For alginate encapsulation, 35ul of 2% W / V were added into each Eppendorf (n=6). Reticulation occurs after the addition of 100ul of 50mM CaCl2 (Sigma-Aldrich, USA). After 15min of reticulation, a hydrogel is formed and shaped with forceps into a ball-shape. Hydrogelis kept in a solution of 50mM CaCl2 till implantation. The ground, lyophilized ECM wasmixed with a fibrin or alginate, and imaged as described above, before and after 6 weeks of ectopic implantation in mice. Results:Combining our ground ECM with fibrin and alginate resulted in scaffolds similar to thelyophilized ECM that was not ground, as revealed by the macroscopic picture (A). Such, confirming that a 3D structure similar to cell-produced ECM can be obtained.Thus demonstrating that the grinding process do not disturb the osteoinductiveproperties of the ECM. After six weeks of implantation, uCT radiographs (B) of all samples showed the formation of smooth mineralized tissue principally localized at the periphery of the implants. The inside of every implant was extensively remodelled, resulting in what can be interpreted as interconnected mineralized bone trabeculae embedding bone marrow elements. Those results were confirmed by Safranin-O staining (C) after paraffin embedding. Each sample presented a mature cortical bonelayer at the periphery and a trabecular structure in the inner part.P7250PC0035 Conclusion:The lyophilized and ground ECM can be successfully combined with a hydrogelsubstrate, such as fibrin or alginate, to create an osteoinductive solution, as demonstrated by the formation of bone in each condition following ectopic implantation in mice. Example 5: Optimization of Fibrinogen and Thrombin Concentrations for Successful Gelation and Injectability of Hydrogel Composites Aim: Determine the fibrinogen and thrombin concentration intervals for successful gelation. Material and Methods:Different concentrations of fibrinogen (5, 7, 9, 11, 13 and 15 mg / ml) were mixed withdifferent concentrations of thrombin (0.5, 1, 1.5, 2 and 2.5 U / ml) and gelation was determined using the inverted vial method. Results: Figure 5A demonstrates an example of successful gelation based on the inverted vialmethod. Figure 5B demonstrates an example of a successful hydrogel. For successfulinjectability of ground lyophilized cartilage tissue combined with fibrinogen andthrombin, 7, 9, 11, 13 or 15 mg / ml fibrinogen, and 1 or 1.5 U / ml thrombin could be used in the mixture (Figure 5C). Conclusion:Successful injectability of ground lyophilized ECM combined with fibrinogen andthrombin was obtained with 7-15 mg / ml fibrinogen and 1-1.5 U / ml thrombin in themixture (Figure 5C).P7250PC0036Example 6: Generation of Humanized Bones via Injectable OssiGel Mixed withHuman Cells Aim:Investigate the bones formed after subcutaneous injection with the ground lyophilizedextracellular matrix (ECM) mixed with fibrin hydrogel and human mesenchymal stemcells (hMSCS). Material and Methods: A human mesenchymal stem cell line, MSOD-B, was seeded on 3D porous collagen scaffolds as described above. In short, the cells on the scaffolds were exposed to chondrogenic factors, for 3 weeks. After 3 weeks of in vitro differentiation, the resulting human cartilage tissue was lyophilized. The lyophilized cartilage tissue was furtherground in order to obtain a fine tissue powder. The ground, lyophilized ECM was mixedwith a fibrin hydrogel. The ground lyophilized ECM with fibrin hydrogel was furthermixed with human mesenchymal stem cells (hMSCS) and subcutaneously injected intomice. After 6 weeks the resulting bones were explanted and imaged on a ZeissAxiophot microscope; and with the U-CT system (MILabs, The Netherlands) using a tungsten x-ray source at 50 kV and 0.21 mA. Results The radiograph of a bone created from hMSCS after being injected subcutaneously with OssiGel after 6-weeks (Figure 6A). The safranin-O staining of the bone shows cortical bone surrounding cartilage, marrow, and trabecular structures derived from human cells (Figure 6B). Furthermore, the safranin-O staining shows cartilage (Figure 6C) and marrow cavities (Figure 6D) within the bone. Conclusion:Subcutaneous injection of mice with ground lyophilized ECM mixed with fibrin hydrogeland hMSCS results in formation of bones showing cortical bone surrounding cartilage,marrow, trabecular structures derived from human cells, cartilage and marrow cavities,after 6 weeks in vivo.P7250PC0037Example 7: Recapitulating bone marrow specific disease: Engraftment of healthyand leukemic human blood cells with OssiGel Aim: Investigate the engraftment of healthy and leukemic human blood cells in the bonesformed after subcutaneous injection with the ground lyophilized extracellular matrix(ECM) tissue mixed with fibrin hydrogel. Material and Methods: A human mesenchymal stem cell line, MSOD-B, was seeded on 3D porous collagen scaffolds as described above. In short, the cells on the scaffolds were exposed to chondrogenic factors, for 3 weeks. After 3 weeks of in vitro differentiation, the resulting human cartilage tissue was lyophilized. The lyophilized cartilage tissue was further ground in order to obtain a fine tissue powder. The ground, lyophilized cartilage was mixed with a fibrin hydrogel. The ground lyophilized cartilage mixed with fibrin hydrogelwas further mixed with CD45+ / CD3- acute myeloid leukemia (AML) cancer cells fromthe blood or bone marrow of AML patients, or human mesenchymal stem cells from ahealthy-donor bone marrow sample or an AML patient bone marrow / biopsy sample (hMSCS), and subcutaneously injected into mice. After 4 weeks, the healthy donorhematopoietic cells or the leukemic cells CD45+ / CD3- from the same AMLpatientrespectively,were directly administered into the ectopically- formed bones orsystemically administered (via teil vein injection). At the end of the experiment, to analyse normal hematopoietic or leukemic engraftment, cells from various tissues were collected, processed, and counted, followed by flow cytometry screening for human CD45 and specific myeloid and lymphoid cell markers, with results expressed as thepercentage of human CD45-positive cells. The explanted bones were visuallyexamined and imaged using a high-resolution mobile digital camera under consistent lighting conditions to ensure uniformity in the analysis. Bone structure was analyzed with µCT radiograph performed with the U-CT system (MILabs). The experimental setup is shown in figure 7A. Results:Healthy donor bone marrow cells mixed with Ossigel could form bone with persistingbone and bone marrow elements post 8 weeks in vivo (trabecular bone, humanmesenchymal cells, adipocytes and blood vessels (Figure 7B). AML cells derived fromP7250PC0038 patients (n=7) engrafted better in our produced humanized bones compared to the gold standard, where AML cells from patients are transplanted directly into mice femurs (Figure 7C). AML engrafted bones showed size heterogeneity (Figure 7D). Different behavior was observed in bones engrafted with AML (Figure 7E, top image), as compared to non-engrafted bones (Figure 7E, bottom). Apart from bone elements (cortical and trabecular bone), AML-derived MSC and leukemic cells could be visualized with a confocal microscope 18 weeks post transplantation and quantified or sorted by flow cytometry / fluorescent activated cell sorting (FACS) (Figure F-H). Conclusion: The bones formed after subcutaneous injection with the ground lyophilized cartilage tissue mixed with fibrin hydrogel allow robust engraftment of a variety of healthy andleukemic human bone marrow or blood cells, for the fundamental study of theirbehaviors or development, such as proliferation, differentiation, heterogeneity andmalignant transformation.Example 8: Generation of ectopic in-vivo bone via OssiGel mixed with prostatecancer patients derived mesenchymal stem cells (Pr-MSC). Aim: Investigate the potential generation of bone following subcutaneous injection with the ground lyophilized extracellular matrix (ECM) tissue mixed with fibrin hydrogel and mesenchymal stem cells derived from prostate cancer patients to study bonemetastatic cancers.Material and Methods: A human mesenchymal stem cell line, MSOD-B, was seeded on 3D porous collagen scaffolds as described above. In short, the cells on the scaffolds were exposed to chondrogenic factors, for 3 weeks. After 3 weeks of in vitro differentiation, the resulting human cartilage tissue was lyophilized. The lyophilized cartilage tissue was further ground in order to obtain a fine tissue powder. The ground, lyophilized cartilage was mixed with a fibrin hydrogel. The ground lyophilized cartilage mixed with fibrin hydrogel was further mixed with human mesenchymal stem cells from prostate cancer patients (prostatectomies) and subcutaneously injected into mice. After 4 weeks, theexperiment ended and the formed bones explanted, were visually examined andimaged using a high-resolution mobile digital camera under consistent lightingP7250PC0039 conditions to ensure uniformity in the analysis. Bone structure was analyzed with µCT radiograph performed with the U-CT system (MILabs). Immunofluorescent staining was performed under a confocal microscope (Leica stellaris 5). Results: Prostate cancer mesenchymal stem cells mixed with Ossigel could form bone with persisting bone and bone marrow elements post 4 weeks in vivo (cortical bone with osteoblastic lining, human mesenchymal stromal cells and their progeny, and bloodvessels (Figure 8A-B). Microcomputed tomography showed typical bone and bonemarrow spaces (trabeculae) (Figure 8C).Conclusion:The ground lyophilized cartilage tissue mixed with fibrin hydrogel allows robust boneformation of prostate cancer derived MSCs. Further administration of the respective patients’ prostate cancer cells in the generated bones would allow the systematic study of bone metastasized prostate cancer development and therapeutic intervention.Example 9: Generation of ectopic in-vivo bone via OssiGel mixed with humaninduced pluripotent stem cells (iPSC) and induced pluripotent stem cell derived mesenchymal stem cells (iPS-MSC). Aim: Investigate the potential generation of bone following subcutaneous injection with the ground lyophilized extracellular matrix (ECM) tissue mixed with fibrin hydrogel and human induced pluripotent stem cells or human differentiated MSC from induced pluripotent stem cell sources. Material and Methods: A human mesenchymal stem cell line, MSOD-B, was seeded on 3D porous collagen scaffolds as described above. In short, the cells on the scaffolds were exposed to chondrogenic factors, for 3 weeks. After 3 weeks of in vitro differentiation, the resulting human cartilage tissue was lyophilized. The lyophilized cartilage tissue was further ground in order to obtain a fine tissue powder. The ground, lyophilized cartilage was mixed with a fibrin hydrogel. The ground lyophilized cartilage mixed with fibrin hydrogel was further mixed with human induced pluripotent stem cells (iPSC) or induced pluripotent stem cell derived mesenchymal stem cells (iPS-MSC) and subcutaneouslyP7250PC0040 injected into mice. After 8 and 6 weeks respectively, the experiments ended and theformed bones explanted, were visually examined and imaged using a high-resolutionmobile digital camera under consistent lighting conditions to ensure uniformity in the analysis. Immunofluorescent staining was performed under a confocal microscope (Leica stellaris 5). Results: Both iPSC and iPS-MSC mixed with Ossigel could form bone with persisting bone and bone marrow elements post 8 and 6 weeks in vivo (cortical bone with osteoblastic lining, human mesenchymal stromal cells and their progeny, and blood vessels (Figure 9A-B). Conclusion: Subcutaneous injection with the ground lyophilized cartilage tissue mixed with fibrin hydrogel allow successful bone formation from human iPSC as well as iPSC-derived MSC sources. These are representing unlimited sources of human cells which could be used in clinical setting as universal grafts or allow future therapies where bone forming cells from the patient are not available or are damaged.Example 10: Chondrogenic differentiation of human bone marrow-derivedmesenchymal stem cells (hBM-MSCs) and human mesenchymal stem cells derived from induced pluripotent stem cells (hiPSCs-MSCs) using OssiGel. Aim: To evaluate whether OssiGel improves or restores the chondrogenic differentiation potential of human bone marrow-derived mesenchymal stem cells (hBM-MSCs), compared to standard culture conditions using a collagen sponge scaffold. Material and Methods: HBM-MSCs and hiPSCs-MSCs were expanded under standard culture conditions and seeded (2x106cells / scaffold) in different 3D culture environments: Control conditions where the cells were cultured in collagen sponges and within the OssiGel, were cells are encapsulated within the OssiGel. Both groups were cultured under chondrogenic differentiation conditions for a defined period (3-weeks). Histological analysis was performed on fixed samples using Safranin O / Fast Green staining to evaluate theP7250PC0041 production of cartilaginous extracellular matrix (ECM), with red indicating glycosaminoglycan (GAG) deposition, indicating positive chondrogenic differentiation. Results: Histological analysis revealed enhanced Safranin-O positive red staining throughout theOssiGel group compared to the control counterpart, indicating an increase of GAG-richcartilage matrix deposition (Figure 10A).Conclusion: OssiGel provides a superior microenvironment for chondrogenic differentiation of hMSCs, either restoring or enhancing their cartilage-forming potential compared to traditional collagen sponge scaffolds. Example 11: Evaluation of the dose-dependent effect of hBM-MSCs encapsulated in OssiGel on ectopic bone formation. Aim: To assess the impact of varying doses of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) encapsulated in OssiGel on bone formation in vivo. Material and Methods: Different concentration of cells (hBM-MSCs at doses of 0, 100.000, 200.000 and 1.000.000 cells) were encapsulated within the Ossigel, and ectopically implanted for 6- weeks in the back of Nude Athymic mice (Crl:NU(NCr)-Foxn1nu). After explantation, bone formation was evaluated via: A. Macroscopic picture of explanted bones for each group after 6-weeks in vivo. B. uCT representative 3D micrographs of explanted bones for each group after 6-weeks in vivo. C. BV / TV% quantification with uCT of explanted bones for each group after 6-weeks in vivo. Results: Similar gross morphology was observed for each condition indicating that OssiGel alonepossess intrinsic osteoinductive potential, regardless of cell dose (Figure 11A). µCTrevealed internal mineralization across all groups, including the acellular control,confirming the osteoinductive nature of the OssiGel scaffold (Figure 11B). However,P7250PC0042 increased doses of hBM-MSCs appeared to enhance the trabecular-like architecture, suggesting an influence on internal bone patterning and possible development ofmarrow-like cavities (Figure 11B). Quantitative analysis showed no statisticallysignificant differences in BV / TV% between the groups (ns), indicating that totalmineralized volume was not substantially altered by cell dose (Figure 11C). OssiGelenables efficient bone formation with minimal cell input. Conclusion: OssiGel supports ectopic bone formation even in the absence of cells, confirming its intrinsic osteoinductive properties. While increasing the dose of hBM-MSCs did not significantly alter the total bone volume (BV / TV%), it influenced the internalmicroarchitecture, promoting the development of trabecular bone-like structures andmarrow-like cavities, especially at higher doses. Importantly, even low cell numbers (asfew as 100,000 cells) were sufficient to induce mineralization, suggesting that cellencapsulation within OssiGel enabling efficient bone formation with minimal cell input.Example 12: Demonstration of chimeric bone formation derived fromencapsulated hBM-MSCs using OssiGel. Aim: To determine whether human bone marrow-derived mesenchymal stem cells (hBM-MSCs) encapsulated in OssiGel contribute directly to bone tissue formation in vivo, andto characterize the human cell contribution to the ectopically formed bone. Material and Methods: 1.000.000 hBM-MSCs were encapsulated within the OssiGel biomaterial and ectopically implanted into the dorsal subcutaneous space of immunocompromised Nude Athymic mice (Crl:NU(NCr)-Foxn1^nu). After 6 weeks, explants were harvested and processedfor immunofluorescence staining. Sections were labeled with HuNu (human nucleimarker, specific to human cells), Osteocalcin (marker of mature osteoblasts andosteocytes) and DAPI (nuclear DNA stain labeling all species).Fluorescent confocal microscopy was used to assess spatial localization and cellular identity within the ossicle structures.P7250PC0043 Results:Representative confocal images highlighting the chimeric origin of the humanizedossicles derived of the implantation of 1.000.000 hBM-MSCs with Ossigel (Figure 12A).Conclusion: Confocal imaging revealed HuNu-positive cells integrated within the newly formed bonetissue, confirming the human origin of a subset of the resident cells. These HuNu+ cellsco-localized with osteocalcin, indicating their differentiation into mature bone-forming cells. DAPI staining confirmed the overall cellularity and allowed identification of both mouse and human nuclei.Example 13: Proteins found by mass spectrometry on the grounded extracellularmatrix. Aim: To gain deeper insight into the composition of the extracellular matrix (ECM) of OssiGel,a comprehensive protein characterization was performed using high-resolution massspectrometry. Material and Methods: Human mesenchymal stem cells (MSOD-B line) were cultured on three-dimensionalporous collagen scaffolds as previously described. Briefly, cells were exposed tochondrogenic differentiation medium for three weeks. Following this period, theengineered cartilage tissue was lyophilized and mechanically pulverized using liquidnitrogen and a mortar and pestle to obtain a fine powder. Protein extraction wasperformed by adding 15 volumes (v / w) of a chaotropic buffer composed of 4 M guanidinehydrochloride, 50 mM sodium acetate, 100 mM 6-aminocaproic acid, 5 mM benzamidine, and 5 mM N-ethylmaleimide, adjusted to pH 5.8. The extraction proceeded for 24 hoursat 4 °C on an orbital shaker. Samples were centrifuged at 13,200 × g for 30 minutes, andproteins were precipitated with nine volumes of ethanol overnight at 4 °C. Thisprecipitation step was repeated, and the resulting protein pellets were dissolved in 0.1 Mammonium bicarbonate buffer at pH 8.5. Protein digestion was carried out by incubating samples with 1 mg of sequencing-gradetrypsin (Promega) at 37 °C for 16 hours on a shaker. Peptide concentrations werequantified using a colorimetric assay (Pierce). To remove peptides associated withP7250PC0044glycosaminoglycan (GAG) chains, samples were diluted 1:1 with 1 M NaCl and filteredusing Nanosep 30 K ultrafiltration units (PALL). Desalting was performed using C18 spincolumns (UltraMicro Spin, Harvard Apparatus), and peptides were eluted with 50%acetonitrile in 0.1% formic acid. Samples were dried under vacuum (SpeedVac) andreconstituted in 0.1% formic acid for mass spectrometry analysis. Mass spectrometry was conducted on an Orbitrap Fusion mass spectrometer equipped with an EASY-Spray ion source and coupled to an EASY-nLC 1000 liquidchromatography system (Thermo Fisher Scientific). Data acquisition was performed inpositive data-dependent mode. Protein identification was conducted using the UniProthuman database (UP5640, release 2018-10) via Proteome Discoverer version 2.2(Thermo Scientific). Quantification of protein abundance was achieved through label-free analysis by summing the peak areas of multiple unique peptides assigned to each protein. Results: Mass spectrometry analysis revealed a highly complex extracellular matrix (ECM),comprising approximately 2,009 proteins. From this dataset, a curated subset of 73proteins characteristic of cartilaginous matrices was identified and categorized based ontheir functional roles within the ECM. These proteins were grouped into five maincategories: (1) Structural ECM proteins, (2) Proteoglycans and small leucine-richproteoglycans (SLRPs), (3) ECM remodeling enzymes, (4) Bone and cartilage formationregulators, and (5) Growth factors.OssiGel's ECM was found to be rich in collagens, with a wide array of fibrillar (COL1A2,COL3A1, COL5A1 / 2, COL11A1 / 2), fibril-associated collagens with interrupted triplehelices (FACITs such as COL12A1, COL14A1), network-forming (COL6A1 / 2 / 3,COL10A1), and cartilage-specific types (COL2A1, COL11A1).Structural ECM glycoproteins (Table 1) included fibronectin (FN1), tenascin-C (TNC),laminins (LAMB1, LAMC1), vitronectin (VTN), thrombospondin-1 (THBS1), andmembers of the fibulin family (FBLN1, FBLN2, FBLN5), contributing to matrixorganization and cell-matrix interactions. EMILIN-1 was also notably present, indicativeof elastic fiber network involvement.P7250PC0045Among proteoglycans and small leucine-rich proteoglycans (SLRPs) (Table 2), majorconstituents included decorin (DCN), biglycan (BGN), aggrecan (ACAN), lumican (LUM),prolargin (PRELP), and fibromodulin (FMOD), which play critical roles in collagenfibrillogenesis and matrix hydration. Notably, versican (VCAN) and osteoglycin (OGN)were detected, linking the construct to cartilage-like functionality.Key ECM-remodeling enzymes (Table 3) were identified, including matrixmetalloproteinases (MMP2, MMP14), lysyl oxidase (LOX), and cathepsin D (CTSD), aswell as serpin H1 (HSP47), a collagen-specific chaperone, and transglutaminase 2(TGM2), suggesting active matrix maturation and stabilization processes.Proteins associated with cartilage and bone development (Tale 4) such as cartilageoligomeric matrix protein (COMP), periostin (POSTN), and CRTAP were highlyrepresented. Growth and signaling molecules (Table 5) including TGFBI, BMP2,IGFBP3, and CTGF were also detected, indicating an active signaling environmentcompatible with endochondral ossification and tissue regeneration. Table 1: Structural ECM Proteins Protein Name Gene Symbol Abundance (a.u.,scientific notation) Fibronectin FN1 4.68 × 10¹¹Tenascin TNC 2.47 × 10¹¹Collagen alpha-3(VI) chain COL6A3 9.64 × 10¹⁰Collagen alpha-1(XII)COL12A1 5.35 × 10¹⁰chain Collagen alpha-2(I) chain COL1A2 6.82 × 10¹⁰Collagen alpha-1(III) chain COL3A1 8.11 × 10⁹Collagen alpha-1(VI) chain COL6A1 2.85 × 10¹⁰Collagen alpha-2(VI) chain COL6A2 2.08 × 10¹⁰Collagen alpha-1(V) chain COL5A1 6.97 × 10⁹Collagen alpha-2(V) chain COL5A2 9.06 × 10⁹Collagen alpha-1(II) chain COL2A1 1.49 × 10¹⁰Collagen alpha-1(XI) chain COL11A1 1.49 × 10⁹Collagen alpha-2(XI) chain COL11A2 3.09 × 10⁸Collagen alpha-1(VII)COL7A1 6.78 × 10⁸chain Collagen alpha-1(X) chain COL10A1 2.13 × 10⁹Collagen alpha-1(XIV)COL14A1 2.49 × 10⁷chain Collagen alpha-1(XVIII)COL18A1 3.36 × 10⁶chain Laminin subunit beta-1 LAMB1 4.07 × 10⁸Laminin subunit gamma-1 LAMC1 2.77 × 10⁸P7250PC0046 Laminin subunit beta-2 LAMB2 2.65 × 10⁷Vitronectin VTN 1.42 × 10⁷Thrombospondin-1 THBS1 3.89 × 10¹⁰Fibrillin-1 FBN1 1.45 × 10⁹Fibulin-1 FBLN1 1.63 × 10⁸Fibulin-2 FBLN2 2.05 × 10⁷Fibulin-5 FBLN5 9.49 × 10⁶EMILIN-1 EMILIN1 2.22 × 10⁹Perlecan HSPG2 1.42 × 10¹⁰Nidogen-2 NID2 2.30 x 10⁸Table 2: Proteoglycans & SLRPs Protein Name Gene Symbol Abundance (a.u.,scientific notation) Decorin DCN 1.09 × 10¹⁰Biglycan BGN 5.20 × 10⁹Aggrecan core protein ACAN 4.44 × 10⁹Lumican LUM 8.29 × 10⁹Mimecan (Osteoglycin) OGN 7.24 × 10⁸Versican VCAN 1.32 × 10⁸Fibromodulin FMOD 2.16 × 10⁹Prolargin PRELP 8.45 × 10⁹Osteomodulin OMD 7.10 × 10⁸Chondroadherin CHAD 1.02 × 10⁸Serglycin SRGN 5.28 × 10⁷Hyaluronan andHAPLN1 3.41 x 10⁹proteoglycan link protein 1 Table 3: ECM Remodeling Enzymes Protein Name Gene Symbol Abundance (a.u.,scientific notation) Matrix metalloproteinase-2 MMP2 1.29 × 10⁹Matrix metalloproteinase-MMP14 2.26 × 10⁷14 Serpin H1 (HSP47) SERPINH1 1.59 × 10¹⁰Lysyl oxidase LOX 4.29 × 10⁸Cathepsin D CTSD 2.77 × 10⁹SPARC (osteonectin) SPARC 8.87 × 10⁸Cartilage oligomeric matrixCOMP 1.08 × 10⁹protein Peroxidasin PXDN 1.55 × 10⁸Protein-glutamine gamma-TGM2 6.36 × 10⁶glutamyltransferase 2 Transglutaminase 2 TGM2 6.36 x 10⁶P7250PC0047 Table 4: Bone & Cartilage Formation Regulators Protein Name Gene Symbol Abundance (a.u.,scientific notation) Cartilage-associatedCRTAP 1.03 × 10⁹protein Bone morphogeneticBMP2 7.65 × 10⁸protein 2 Bone morphogeneticBMP1 1.75 × 10⁷protein 1 Periostin POSTN 2.22 × 10⁹Chondroadherin CHAD 1.02 x 10⁸Table 5: Growth Factors Protein Name Gene Symbol Abundance (a.u.,scientific notation) Transforming growthTGFBI 8.45 × 10¹⁰factor-beta-induced protein ig-h3 Bone morphogeneticBMP2 7.65 × 10⁸protein 2 Connective tissue growthCTGF 1.64 × 10⁸factor Follistatin FST 1.95 × 10⁶Angiopoietin-relatedANGPTL4 1.82 × 10⁸protein 4 Insulin-like growth factor-IGFBP3 5.19 × 10⁹binding protein 3 Midkine MDK 1.59 × 10⁷Conclusion: The proteomic profile of OssiGel demonstrates the presence of a rich and diverse ECMcomposition similar to cartilage with a complex matrix, rich in fibrillar collagens,glycoproteins, proteoglycans, and remodelling enzymes. The results confirm that theECM derived from chondrogenic differentiation of MSOD-B resulted in an ECM rich in both cartilage-associated (COL2A1, aggrecan) and bone-associated (COL1A2) proteins. This hybrid matrix profile is characteristic of hypertrophic cartilage, which naturally transitions toward bone formation during endochondral ossification. Notably, the strong presence of BMP2 further highlights the scaffold’s potential for supportingbone formation. The hypertrophic cartilage nature of OssiGel, positions the material asa biomaterial uniquely suited for supporting endochondral ossification. The rich contentof proteoglycans and glycoproteins makes OssiGel an excellent material for supporting cell attachment, proliferation, and differentiation, making it attractive for various in vitroand in vivo applications.P7250PC0048 Items 1. A ground extracellular matrix (ECM) produced by a human stem cell line.2. A composition comprising:a. the ECM according to item 1, andb. a gel-like substrate or a precursor thereof.3. The composition according to item 2, wherein the composition inducesformation of bone and / or bone marrow in-vivo.4. The composition according to item 2, wherein the composition inducesformation of bone and / or bone marrow in-vitro.5. The composition according to any one of items 2 to 4, wherein the ground ECMhas a particle size ranging from 1 to 1000 µm, such as ranging from 1 to 100 µm, such as ranging from 1 to 200 µm, ranging from 1 to 300 µm, ranging from 1 to 400 µm, ranging from 1 to 500 µm, ranging from 1 to 600 µm, ranging from 1 to 700 µm, ranging from 1 to 800 µm, ranging from 1 to 900 µm, such as ranging from 900 to 1000 µm, such as ranging from 800 to 1000 µm, such as ranging from 700 to 1000 µm, such as ranging from 600 to 1000 µm, such as ranging from 500 to 1000 µm, such as ranging from 400 to 1000 µm, such as ranging from 300 to 1000 µm, such as ranging from 200 to 1000 µm, such as ranging from 100 to 1000 µm. 6. The composition according to any one of items 2 to 5, wherein the human stemcell line is a human mesenchymal stem cell line. 7. The composition according to any one of items 2 to 6, wherein the humanmesenchymal stem cell line is a stem cell line overexpressing Bone Morphogenetic Protein-2 (BMP-2).8. The composition according to any one of items 2 to 7, wherein the stem cell lineoverexpressing BMP-2 is the Mesenchymal Sword of Damocles – BMP2(MSOD-B) cell line.P7250PC00499. The composition according to any one of items 2 to 8, wherein the precursorcomprises non-dissolved components of the gel-like substrate.10. The composition according to any one of items 2 to 9, wherein the componentsare selected from the group consisting of fibrinogen, thrombin, alginate and / or polyethylene glycols.11. The composition according to any one of items 2 to 10, wherein the gel-likesubstrate or precursor thereof is synthetic and / or naturally occurring.12. The composition according to any one of items 2 to 11, wherein the naturallyoccurring gel-like substrate or precursor thereof comprises fibrinogen and / orthrombin.13. The composition according to any one of items 2 to 12, wherein the gel-likesubstrate or precursor thereof comprises fibrinogen.14. The composition according to any one of items 2 to 13, wherein thrombin isadded to the gel-like substrate or precursor thereof comprising fibrinogen toinduce cross-linking and / or polymerization.15. The composition according to any one of items 2 to 14, wherein thrombin isadded to the gel-like substrate or precursor thereof comprising fibrinogen priorto in vivo injection of the composition, such as 10 minutes, such as 9 minutes, such as 8 minutes, such as 7 minutes, such as 6 minutes, such as 5 minutes, such as 4 minutes, such as 3 minutes, such as 2 minutes, such as 1 minute prior to in vivo injection.16. The composition according to any one of items 2 to 15, wherein thrombin isadded to the gel-like substrate or precursor thereof comprising fibrinogen 5minutes prior to in vivo injection of the composition.17. The composition according to any one of items 2 to 16, wherein the naturallyoccurring gel-like substrate or precursor thereof comprises alginate.P7250PC005018. The composition according to any one of items 2 to 17, wherein the syntheticgel-like substrate or precursor thereof comprises polyethylene glycols.19. The composition according to any one of items 2 to 18, wherein the syntheticgel-like substrate or precursor thereof comprises 8 to 40 % polyethyleneglycols.20. The composition according to any one of items 2 to 19, wherein the gel-likesubstrate or precursor thereof comprises at least 5 mg / ml fibrinogen, such a 6mg / ml, such as 7 mg / ml, such as 8 mg / ml, such as 9 mg / ml, such as 10 mg / ml,such as 11 mg / ml, such as 12 mg / ml, such as 13 mg / ml, such as 14 mg / ml, such as 15 mg / ml, such as 16 mg / ml, such as 17 mg / ml, such as 18 mg / ml,such as 19 mg / ml, such as 20 mg / ml.21. The composition according to any one of items 2 to 20, wherein the gel-likesubstrate or precursor thereof comprises 7 to 15 mg / ml fibrinogen.22. The composition according to any one of items 2 to 21, wherein the gel-likesubstrate or precursor thereof comprises at least 0.5 U / ml thrombin, such as 0.6U / ml, such as 0.7 U / ml, such as 0.8 U / ml, such as 0.9 U / ml, such as 1 U / ml, such as 1.1 U / ml, such as 1.2 U / ml, such as 1.3 U / ml, such as 1.4 U / ml, such as 1.5 U / ml, such as 1.6, U / ml, such as 1.7, U / ml, such as 1.8, U / ml, such as 1.9, U / ml, such as 2 U / ml, such as 2.1 U / ml, such as 2.2 U / ml, such as 2.3 U / ml, such as 2.4 U / ml, such as 2.5 U / ml, such as 2.6, U / ml, such as 2.7, U / ml, such as 2.8, U / ml, such as 2.9, U / ml, such as 3 U / ml.23. The composition according to any one of items 2 to 22, wherein the gel-likesubstrate or precursor thereof comprises 1 to 1.5 U / ml thrombin.24. The composition according to any one of items 2 to 23, wherein the compositioncomprises at least 0.1 mg ground ECM, such as 0.2 mg, such as 0.3 mg, such as 0.4 mg, such as 0.5 mg, such as 0.5 mg, such as 0.6 mg, such as 0.7 mg, such as 0.8 mg, such as 0.9 mg, such as 1 mg, such as 2 mg, such as 3 mg, such as 4 mg, such as 5 mg , such as 6 mg, such as 7 mg, such as 8 mg, such as 9 mg, such as 10 mg.P7250PC005125. The composition according to any one of items 2 to 24, wherein the compositioncomprises 1 mg ground ECM.26. The composition according to any one of items 2 to 25, wherein the compositioncomprises 1 mg ground ECM, 7.5-wt % human fibrin and 0.9 U / mL humanthrombin.27. The composition according to any one of items 2 to 26, wherein the compositionfurther comprises cells.28. The composition according to any one of items 2 to 27, wherein the cells arehuman cells.29. The composition according to any one of items 2 to 28, wherein the human cellsare human stem cells.30. The composition according to any one of items 2 to 29, wherein the humanstem cells are human mesenchymal stem cells.31. The composition according to any one of items 2 to 30, wherein the human cellsare human cancer cells.32. The composition according to any one of items 2 to 31, wherein the cancer cellsare leukemia cells.33. The composition according to any one of items 2 to 32, wherein the cancer cellsare solid cancer cells.34. The composition according to any one of items 2 to 33, wherein the leukemiacells are selected from the group consisting of acute myeloid leukemia (AML) cells, acute lymphocytic leukemia (ALL) cells, chronic lymphocytic leukemia(CLL) cells, and chronic myeloid leukemia (CML) cells.35. The composition according to any one of items 2 to 34, wherein the solid cancercells are selected from the group consisting of bone cancer cells, prostate cancer cells, breast cancer cells, and neuroblastoma cells.P7250PC005236. The composition according to any one of items 2 to 35, wherein the leukemiacells are AML cells.37. The composition according to any one of items 2 to 36, wherein the AML cellsare mononucleated cells, such as CD45+ / CD3- AML cells.38. The composition according to any one of items 2 to 37, wherein the AML cellsare isolated from the blood or bone marrow of AML patients.39. The composition according to any one of items 2 to 38, wherein the compositionfurther comprises at least 2 µg / ml glycosaminoglycans, such as 3 µg / ml, suchas 4 µg / ml, such as 5 µg / ml, such as 6 µg / ml, such as 7 µg / ml, such as 8 µg / ml,such as 9 µg / ml, such as 10 µg / ml, such as 11 µg / ml, such as 12 µg / ml, suchas 13 µg / ml, such as 14 µg / ml, such as 15 µg / ml, such as 16 µg / ml, such as 17µg / ml, such as 18 µg / ml, such as 19 µg / ml, such as 20 µg / ml.40. The composition according to any one of items 2 to 39, wherein the compositionfurther comprises at least 10 µg / ml Bone Morphogenetic Protein-2 (BMP-2),such as 15 µg / ml, such as 20 µg / ml, such as 25 µg / ml, such as 30 µg / ml, suchas 35 µg / ml, such as 40 µg / ml such as 45 µg / ml, such as 50 µg / ml, such as 55µg / ml, such as 60 µg / ml.41. The composition according to any one of items 2 to 40, wherein the groundECM and the gel-like substrate or precursor thereof are mixed with each other.42. The composition according to any one of items 2 to 41, further comprising cellculture media.43. The composition according to any one of items 2 to 42, wherein the cell culturemedia is Dulbecco’s modified eagle’s medium (DMEM).44. A kit comprising the composition according to any one of the preceding items,phosphate-buffered saline, cell culture media, and instructions for use.45. The kit according to item 44, further comprising thrombin.46. The kit according to any one of items 44 to 45, further comprising cells.P7250PC005347. A method of manufacturing the composition according to any one of items 2 to43, the method comprising the following steps: a. culturing a stem cell line in vitro;b. exposing the stem cell line to differentiation factors, therebydifferentiating said stem cell line into mesenchymal lineage cell types;c. culturing the differentiated cells of step b, thereby producing anextracellular matrix (ECM); d. lyophilizing the cells comprised in the ECM of step c, thereby obtainingsaid ECM; e. grinding the ECM of step d, thereby obtaining a ground ECM;and / or f. mixing the ground ECM with a gel-like substrate or precursor thereof.48. The method according to item 47, wherein the stem cell line is cultured on 2Dculture plates.49. The method according to any one of items 47 to 48, wherein the stem cell line iscultured on 3D porous scaffolds.50. The method according to any one of items 47 to 49, wherein the 3D porousscaffolds are collagen scaffolds, such as sponges.51. The method according to any one of items 47 to 50, wherein the collagen iscollagen type I.52. The method according to any one of items 47 to 51, wherein the stem cell line isa human stem cell line.53. The method according to any one of items 47 to 52, wherein the human stemcell line is a human mesenchymal stem cell line.54. The method according to any one of the items 47 to 53, wherein the humanmesenchymal stem cell line is a stem cell line overexpressing Bone Morphogenetic Protein-2 (BMP-2).P7250PC005455. The method according to any one of items 47 to 54, wherein the stem cell lineoverexpressing BMP-2 is the Mesenchymal Sword of Damocles – BMP2(MSOD-B) cell line.56. The method according to any one of items 47 to 55, wherein the differentiationfactors are chondrogenic factors.57. The method according to any one of items 47 to 56, wherein the chondrogenicfactors are selected from the group consisting of, transferrin, selenium, ascorbic acid, dexamethasone, TGF-β3, TGF-β1, BMP-2, BMP-7, and BMP-4.58. The method according to item 57, wherein the ascorbic acid concentration is atleast 0.01 mM, such as 0.05 mM, such as 0.1 mM, such as 0.2 mM, such as 0.5 mM.59. The method according to item 57, wherein the dexamethasone concentration isat least 0.01 µM, such as 0.05 µM, such as 0.1 µM, such as 0.5 µM, such as 1µM, such as 1.5 µM, such as 2 µM.60. The method according to item 57, wherein the TGF-β3 concentration is at least1 ng / ml, such as 2 ng / ml, such as 5 ng / ml, such as 10 ng / ml, such as 15 ng / ml, such as 20 ng / ml.61. The method according to any one of items 47 to 60, wherein the stem cell line isexposed to differentiation factors for at least 3 days, such as at least 4 days, such as at least 5 days, such as at least 6 days, such as at least 7 days, suchas at least 8 days, such as at least 9 days, such as at least 10 days, such as atleast 11 days, such as at least 12 days, such as at least 13 days, such as atleast 14 days, such as at least 15 days, such as at least 16 days, such as atleast 17 days, such as at least 18 days, such as at least 19 days, such as atleast 20 days, such as at least 21 days, such as at least 22 days, such as atleast 23 days, such as at least 24 days, such as at least 25 days, such as atleast 26 days, such as at least 27 days, such as at least 28 days, such as atleast 29 days, such as at least 30 days, such as at least 31 days, such as atleast 32 days, such as at least 33 days, such as at least 34 days, such as atleast 35 days.P7250PC005562. The method according to any one of items 47 to 61, wherein the stem cell line isexposed to differentiation factors for at least 1 week, such as at least 2 week, such as at least 3 weeks, such as at least 4 weeks, such as at least 5 weeks.63. The method according to any one of items 47 to 62, wherein the cells arefurther exposed to penicillin-streptomycin-glutamine, sodium pyruvate, and insulin.64. The method according to any one of items 47 to 63, wherein the mesenchymallineage cell types are selected from the group consisting of bone cells, fat cells, cartilage cells and / or muscle cells.65. The method according to any one of items 47 to 64, wherein the cells are rinsedwith PBS before lyophilization.66. The method according to any one of items 47 to 65, wherein the cells are snapfrozen for 5 minutes in liquid nitrogen before lyophilization.67. The method according to any one of items 47 to 66, wherein the cartilage tissueis lyophilized overnight.68. The method according to any one of items 47 to 67, wherein the cartilage tissueis lyophilized at -80°C.69. The method according to any one of items 47 to 68, wherein the cartilagetissue is lyophilized with a pressure of 0.05 mbar.70. The method according to any one of items 47 to 69, wherein the ECM is groundwith the CryoMill.71. The method according to any one of items 47 to 70, wherein the ECM is groundin a grinding jar.72. The method according to any one of items 47 to 71, wherein the grinding jarhas a volume of 10 ml.73. The method according to any one of items 47 to 72, wherein the grinding jar isof stainless steel.P7250PC005674. The method according to any one of items 47 to 73, wherein the grinding jarcomprises two 10 mm grinding balls.75. The method according to any one of items 47 to 74, wherein the ECM is groundfor 4 x 2 minutes.76. The method according to any one of items 47 to 75, wherein the ECM is groundat 25 hz.77. The method according to any one of items 47 to 76, wherein the ground ECMhas a particle size ranging from 1 to 1000 µm, such as ranging from 1 to 100 µm, such as ranging from 1 to 200 µm, ranging from 1 to 300 µm, ranging from 1 to 400 µm, ranging from 1 to 500 µm, ranging from 1 to 600 µm, ranging from 1 to 700 µm, ranging from 1 to 800 µm, ranging from 1 to 900 µm, such as ranging from 900 to 1000 µm, such as ranging from 800 to 1000 µm, such as ranging from 700 to 1000 µm, such as ranging from 600 to 1000 µm, such as ranging from 500 to 1000 µm, such as ranging from 400 to 1000 µm, such as ranging from 300 to 1000 µm, such as ranging from 200 to 1000 µm, such as ranging from 100 to 1000 µm.78. The method according to any one of items 47 to 77, wherein the gel-likesubstrate or precursor thereof is synthetic and / or naturally occurring.79. The method according to any one of items 47 to 78, wherein the naturallyoccurring gel-like substrate or precursor thereof comprises fibrinogen and / orthrombin.80. The method according to any one of items 47 to 79, wherein the naturallyoccurring gel-like substrate or precursor thereof comprises alginate.81. The method according to any one of items 47 to 80, wherein the synthetic gel-like substrate or precursor thereof comprises 8 to 40 % polyethylene glycols.82. The method according to any one items 47 to 81, wherein the gel-like substrateor precursor thereof comprises at least 5 mg / ml fibrinogen, such a 6 mg / ml,such as 7 mg / ml, such as 8 mg / ml, such as 9 mg / ml, such as 10 mg / ml, such as11 mg / ml, such as 12 mg / ml, such as 13 mg / ml, such as 14 mg / ml, such as 15P7250PC0057 mg / ml, such as 16 mg / ml, such as 17 mg / ml, such as 18 mg / ml, such as 19mg / ml, such as 20 mg / ml.83. The method according to any one of items 47 to 82, wherein the gel-likesubstrate or precursor thereof comprises 7 to 15 mg / ml fibrinogen.84. The method according to any one of items 47 to 83, wherein the gel-likesubstrate or precursor thereof comprises at least 0.5 U / ml thrombin, such as 0.6U / ml, such as 0.7 U / ml, such as 0.8 U / ml, such as 0.9 U / ml, such as 1 U / ml, such as 1.1 U / ml, such as 1.2 U / ml, such as 1.3 U / ml, such as 1.4 U / ml, such as 1.5 U / ml, such as 1.6, U / ml, such as 1.7, U / ml, such as 1.8, U / ml, such as 1.9, U / ml, such as 2 U / ml, such as 2.1 U / ml, such as 2.2 U / ml, such as 2.3 U / ml, such as 2.4 U / ml, such as 2.5 U / ml, such as 2.6, U / ml, such as 2.7, U / ml, such as 2.8, U / ml, such as 2.9, U / ml, such as 3 U / ml.85. The method according to any one of items 47 to 84, wherein the gel-likesubstrate or precursor thereof comprises 1 to 1.5 U / ml thrombin.86. The method according to any one of items 47 to 85, wherein the compositioncomprises at least 0.1 mg ground ECM, such as 0.2 mg, such as 0.3 mg, such as 0.4 mg, such as 0.5 mg, such as 0.5 mg, such as 0.6 mg, such as 0.7 mg, such as 0.8 mg, such as 0.9 mg, such as 1 mg, such as 2 mg, such as 3 mg, such as 4 mg, such as 5 mg , such as 6 mg, such as 7 mg, such as 8 mg, such as 9 mg, such as 10 mg.87. The method according to any one of items 47 to 86, wherein the compositioncomprises 1 mg ground ECM.88. The method according to any one of items 47 to 87, wherein the compositionfurther comprises at least 2 µg / ml glycosaminoglycans, such as 3 µg / ml, suchas 4 µg / ml, such as 5 µg / ml, such as 6 µg / ml, such as 7 µg / ml, such as 8 µg / ml,such as 9 µg / ml, such as 10 µg / ml, such as 11 µg / ml, such as 12 µg / ml, suchas 13 µg / ml, such as 14 µg / ml, such as 15 µg / ml, such as 16 µg / ml, such as 17µg / ml, such as 18 µg / ml, such as 19 µg / ml, such as 20 µg / ml.89. The method according to any one of items 47 to 88, wherein the compositionfurther comprises at least 10 µg / ml Bone Morphogenetic Protein-2 (BMP-2),P7250PC0058 such as 15 µg / ml, such as 20 µg / ml, such as 25 µg / ml, such as 30 µg / ml, suchas 35 µg / ml, such as 40 µg / ml such as 45 µg / ml, such as 50 µg / ml, such as 55µg / ml, such as 60 µg / ml.90. The method according to any one of items 47 to 89, further comprising cellculture media.91. The method according to any one of items 47 to 90, wherein the cell culturemedia is Dulbecco’s modified eagle’s medium (DMEM).92. An in vivo method for bone formation, the method comprising the followingsteps: a. administering the composition according to any one of items 2 to 43 to asubject; and b. maintaining for at least 1 week, such as 2 weeks, such as 3 weeks.93. An in vivo method for bone and bone marrow tissue formation, the methodcomprising the following steps: a. administering the composition according to any one of items 2 to 43 to asubject; and b. maintaining for at least 2 weeks, such as 3 weeks, 4 weeks, such as 5weeks, such as 6 weeks, such as 7 weeks, such as 8 weeks.94. The method according to any one of items 92 to 93, wherein the subject is amammal.95. The method according to any one of items 92 to 94, wherein the mammal is amouse, rat, rabbit, pig, dog, guinea pig or monkey.96. The method according to any one of items 92 to 95, wherein the administrationin vivo is a subcutaneous injection.97. The method according to any one of items 92 to 96, wherein the administrationin vivo is an implantation.P7250PC005998. The method according to any one of items 92 to 97, wherein at least 1 mm3,such as 2 mm3, such as 3 mm3, such as 4 mm3, such as 5 mm3, such as 6 mm3, such as 7 mm3, such as 8 mm3, such as 9 mm3, such as 10 mm3, such as 11 mm3, such as 12 mm3, such as 13 mm3, such as 14 mm3, such as 15 mm3, such as 16 mm3, such as 17 mm3, such as 18 mm3, such as 19 mm3, such as 20 mm3bone tissue is formed, at least 1 week, such as 2 weeks, such as 3 weeks, such as 4 weeks, such as 5 weeks, such as 6 weeks post-in vivo administration.99. The method according to any one of items 92 to 98, wherein the amount ofbone tissue formation is assessed by microtomography analysis.100. The method according to any one of items 90 to 97, further comprisingadministering cells.101. An in vitro method for bone and / or bone marrow tissue formation, themethod comprising the following steps: a. placing the composition according to any one of items 1 to 43 in culturemedia; b. administering cells;c. incubating for at least 1 week, such as 2 weeks, such as 3 weeks, suchas 4 weeks, such as 5 weeks, such as 6 weeks, such as 7 weeks, such as 8 weeks.102. The method according to any one of items 92 to 101, wherein the cellsare human cells.103. The method according to any one of items 92 to 102, wherein the humancells are human stem cells.104. The method according to any one of items 92 to 103, wherein the humancells are human mesenchymal stem cells.105. The method according to any one of items 92 to 104, wherein the humancells are human cancer cells.P7250PC0060106. The method according to any one of items 92 to 105, wherein the cancercells are leukemia cells.107. The method according to any one of items 92 to 106, wherein the cancercells are solid cancer cells.108. The method according to any one of items 92 to 107, wherein theleukemia cells are selected from the group consisting of acute myeloid leukemia (AML) cells, acute lymphocytic leukemia (ALL) cells, chronic lymphocyticleukemia (CLL) cells, and chronic myeloid leukemia (CML) cells.109. The method according to any one of items 92 to 108, wherein the solidcancer cells are selected from the group consisting of bone cancer cells, prostate cancer cells, breast cancer cells, and neuroblastoma cells.110. The method according to any one of items 92 to 109, wherein theleukemia cells are AML cells.111. The method according to any one of items 92 to 110, wherein humancells are present in the formed bone marrow microenvironment, at least 1 week,such as at least 2 weeks, such as at least 3 weeks, such as at least 4 weeks, such as at least 5 weeks, such as at least 6 weeks post-in vivo implantation.112. A product obtained by the method according to any one of items 92 to111.113. A method for testing a candidate pharmaceutical product, the methodcomprising the following steps: a. establishing bone and bone marrow according to any one of items 92 to111; b. administering pathological cells to the bone and bone marrow of step a,thereby establishing a model of pathological bone and bone marrow; c. assessing the viability and / or cell number of the pathological cells;d. administering a candidate pharmaceutical product;e. assessing the viability and / or cell number of the pathological cells;andP7250PC0061 f. classifying the candidate pharmaceutical product as effective againstpathological cells if the viability and / or cell number is decreased in step e compared to step c.114. The method according to item 113, wherein the candidate pharmaceuticalproduct is a pharmaceutical product against cancer cells.115. The method according to any one of items 113 to 114, wherein thepharmaceutical product is a drug.116. The method according to any one of items 113 to 115, wherein thepharmaceutical product is an agent used in cell therapy or gene therapy.117. A method for identifying treatment responders, the method comprising thefollowing steps: a. establishing bone and bone marrow according to any one of items 92 to111; b. isolating cells from a patient;c. administering the isolated cells from step b) to the bone and bonemarrow of step a), thereby establishing a model of the bone and bone marrow of said patient; d. assessing the viability and / or cell number of the administered cells in thebone marrow; e. administering a pharmaceutical product;f. assessing the viability and / or cell number of the administered cells;and g. classifying the patient as a responder to the pharmaceutical product ifthe viability and / or cell number is decreased in step f) compared to step d.118. The method according to item 117, wherein the cells are cancer cells.119. The method according to any one of items 113 to 118, wherein the cancercells are engrafted leukemia cells and / or engrafted human solid cancer cells.P7250PC0062 120. The method according to any one of items 113 to 119, wherein theleukemia cells are selected from the group consisting of acute myeloid leukemia (AML) cells, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). 121. The method according to any one of items 113 to 120, wherein the solidcancer cells are selected from the group consisting of bone cancer cells, prostate cancer cells, breast cancer cells, and neuroblastoma cells. 122. The method according to any one of items 113 to 121, wherein theleukemia cells are AML cells.123. The composition according to any one of items 1 to 43, for use inmedicine. 124. The composition according to any one of items 1 to 43, for use in amethod of treating cancer. 125. The composition for use according to item 124, wherein the cancer isselected from the group consisting of leukemia, and solid cancer. References S. Pigeot, T. Klein, F. Gullotta, S. J. Dupard, A. GarciaGarcia, A. García-García, S. Prithiviraj, P. Lorenzo, M. Filippi, C. Jaquiery, L. Kouba, M. A. Asnaghi, D. B. Raina, B. Dasen, H. Isaksson, P. Önnerfjord, M. Tägil, A. Bondanza, I. Martin, P. E. Bourgine, Manufacturing of Human Tissues as off-the-Shelf Grafts Programmed to InduceRegeneration. Adv. Mater.2021, 33, 2103737.

Claims

P7250PC0063 Claims 1. A ground extracellular matrix (ECM) produced by a human stem cell line withchondrogenic activity, wherein the human stem cell line is genetically modified to overexpress Bone Morphogenetic Protein-2 (BMP-2).

2. An injectable composition comprising:a. the ECM according to claim 1, andb. a hydrogel or precursor thereof.

3. The composition according to claim 2, wherein the composition inducesformation of bone and / or bone marrow in vivo.

4. The ECM according to claim 1 and / or the composition according to any one ofclaims 2 to 3, wherein the ground ECM has a particle size ranging from 1 to 1000 µm, such as ranging from 1 to 100 µm, such as ranging from 1 to 200 µm, ranging from 1 to 300 µm, ranging from 1 to 400 µm, ranging from 1 to 500 µm, ranging from 1 to 600 µm, ranging from 1 to 700 µm, ranging from 1 to 800 µm, ranging from 1 to 900 µm, such as ranging from 900 to 1000 µm, such as ranging from 800 to 1000 µm, such as ranging from 700 to 1000 µm, such as ranging from 600 to 1000 µm, such as ranging from 500 to 1000 µm, such as ranging from 400 to 1000 µm, such as ranging from 300 to 1000 µm, such as ranging from 200 to 1000 µm, such as ranging from 100 to 1000 µm.

5. The ECM according to claim 1 and / or the composition according to any one ofclaims 2 to 4, wherein the human mesenchymal stem cell line overexpressingBone Morphogenetic Protein-2 (BMP-2), is the Mesenchymal Sword ofDamocles – BMP2 (MSOD-B) cell line.

6. The composition according to any one of claims 2 to 5, wherein the hydrogel orprecursor thereof is naturally occurring and / or synthetic.

7. The composition according to any one of claims 2 to 6, wherein the naturallyoccurring hydrogel or precursor thereof comprises fibrinogen and / or thrombin.P7250PC00648. The composition according to any one of claims 2 to 7, wherein the precursorcomprises non-dissolved components of the hydrogel.

9. The composition according to any one of claims 2 to 8, wherein the componentsare selected from the group consisting of fibrinogen, thrombin, alginate and / or polyethylene glycols.

10. The composition according to any one of claims 2 to 9, wherein the hydrogel orprecursor thereof comprises or consists of fibrinogen.

11. The composition according to any one of claims 2 to 10, wherein thrombin isadded to the hydrogel or precursor thereof to induce cross-linking and / orpolymerization.

12. The composition according to any one of claims 2 to 11, wherein thrombin isadded to the hydrogel or precursor thereof prior to in vivo injection of thecomposition, such as 10 minutes, such as 9 minutes, such as 8 minutes, such as 7 minutes, such as 6 minutes, such as 5 minutes, such as 4 minutes, such as 3 minutes, such as 2 minutes, such as 1 minute prior to in vivo injection.

13. The composition according to any one of claims 2 to 12, wherein thrombin isadded to the hydrogel or precursor thereof 5 minutes prior to in vivo injection ofthe composition.

14. The composition according to any one of claims 2 to 13, wherein the naturallyoccurring hydrogel or precursor thereof comprises or consists of alginate.

15. The composition according to any one of claims 2 to 14, wherein the synthetichydrogel or precursor thereof comprises or consists of polyethylene glycols.

16. The composition according to any one of claims 2 to 15, wherein the synthetichydrogel or precursor thereof comprises 8 to 40 % polyethylene glycols.

17. The composition according to any one of claims 2 to 16, wherein the hydrogelor precursor thereof comprises:P7250PC0065 a. at least 5 mg / ml fibrinogen, such a 6 mg / ml, such as 7 mg / ml, such as 8mg / ml, such as 9 mg / ml, such as 10 mg / ml, such as 11 mg / ml, such as 12 mg / ml, such as 13 mg / ml, such as 14 mg / ml, such as 15 mg / ml, such as 16 mg / ml, such as 17 mg / ml, such as 18 mg / ml, such as 19 mg / ml,such as 20 mg / ml, preferably 7 to 15 mg / ml fibrinogen; b. at least 0.5 U / ml thrombin, such as 0.6 U / ml, such as 0.7 U / ml, such as0.8 U / ml, such as 0.9 U / ml, such as 1 U / ml, such as 1.1 U / ml, such as 1.2 U / ml, such as 1.3 U / ml, such as 1.4 U / ml, such as 1.5 U / ml, such as 1.6, U / ml, such as 1.7, U / ml, such as 1.8, U / ml, such as 1.9, U / ml, suchas 2 U / ml, such as 2.1 U / ml, such as 2.2 U / ml, such as 2.3 U / ml, such as 2.4 U / ml, such as 2.5 U / ml, such as 2.6, U / ml, such as 2.7, U / ml, such as 2.8, U / ml, such as 2.9, U / ml, such as 3 U / ml, c. at least 0.1 mg ground ECM, such as 0.2 mg, such as 0.3 mg, such as0.4 mg, such as 0.5 mg, such as 0.5 mg, such as 0.6 mg, such as 0.7 mg, such as 0.8 mg, such as 0.9 mg, such as 1 mg, such as 2 mg, such as 3 mg, such as 4 mg, such as 5 mg , such as 6 mg, such as 7 mg, such as 8 mg, such as 9 mg, such as 10 mg, preferably 1 mg ground ECM; d. at least 2 µg / ml glycosaminoglycans, such as 3 µg / ml, such as 4 µg / ml,such as 5 µg / ml, such as 6 µg / ml, such as 7 µg / ml, such as 8 µg / ml,such as 9 µg / ml, such as 10 µg / ml, such as 11 µg / ml, such as 12 µg / ml,such as 13 µg / ml, such as 14 µg / ml, such as 15 µg / ml, such as 16µg / ml, such as 17 µg / ml, such as 18 µg / ml, such as 19 µg / ml, such as20 µg / ml; and / or e. at least 10 µg / ml Bone Morphogenetic Protein-2 (BMP-2), such as 15µg / ml, such as 20 µg / ml, such as 25 µg / ml, such as 30 µg / ml, such as35 µg / ml, such as 40 µg / ml such as 45 µg / ml, such as 50 µg / ml, such as55 µg / ml, such as 60 µg / ml.

18. The composition according to any one of claims 2 to 17, wherein the hydrogelor precursor thereof comprises 1 to 1.5 U / ml thrombin.P7250PC006619. The composition according to any one of claims 2 to 18, wherein thecomposition comprises or consists of 1 mg ground ECM, 7.5-wt % human fibrinand 0.9 U / mL human thrombin.

20. The composition according to any one of claims 2 to 19, wherein thecomposition further comprises cells.

21. The composition according to any one of claims 2 to 20, wherein the cells arehuman cells.

22. The composition according to any one of claims 2 to 21, wherein the humancells are human stem cells such as human bone marrow-derived mesenchymalstem cells (hBM-MSCs), and / or human pluripotent stem cells, such as inducedpluripotent stem cells (iPCS).

23. The composition according to any one of claims 2 to 22, wherein the humanstem cells are human mesenchymal stem cells.

24. The composition according to any one of claims 2 to 23, wherein the humancells are human cancer cells.

25. The composition according to any one of claims 2 to 24, wherein the cancercells are leukemia cells.

26. The composition according to any one of claims 2 to 25, wherein the cancercells are solid cancer cells.

27. The composition according to any one of claims 2 to 26, wherein the leukemiacells are selected from the group consisting of acute myeloid leukemia (AML) cells, acute lymphocytic leukemia (ALL) cells, chronic lymphocytic leukemia(CLL) cells, and chronic myeloid leukemia (CML) cells.

28. The composition according to any one of claims 2 to 27, wherein the solidcancer cells are selected from the group consisting of bone cancer cells, prostate cancer cells, breast cancer cells, and neuroblastoma cells.P7250PC006729. The composition according to any one of claims 2 to 28, wherein the leukemiacells are AML cells.

30. The composition according to any one of claims 2 to 29, wherein the AML cellsare mononucleated cells, such as CD45+ / CD3- AML cells.

31. The composition according to any one of claims 2 to 30, wherein the AML cellsare isolated from the blood or bone marrow of AML patients.

32. The composition according to any one of claims 2 to 31, wherein thecomposition further comprises at least 2 µg / ml glycosaminoglycans, such as 3 µg / ml, such as 4 µg / ml, such as 5 µg / ml, such as 6 µg / ml, such as 7 µg / ml,such as 8 µg / ml, such as 9 µg / ml, such as 10 µg / ml, such as 11 µg / ml, such as12 µg / ml, such as 13 µg / ml, such as 14 µg / ml, such as 15 µg / ml, such as 16µg / ml, such as 17 µg / ml, such as 18 µg / ml, such as 19 µg / ml, such as 20 µg / ml.

33. The composition according to any one of claims 2 to 32, wherein thecomposition further comprises at least 10 µg / ml Bone Morphogenetic Protein-2(BMP-2), such as 15 µg / ml, such as 20 µg / ml, such as 25 µg / ml, such as 30µg / ml, such as 35 µg / ml, such as 40 µg / ml such as 45 µg / ml, such as 50 µg / ml,such as 55 µg / ml, such as 60 µg / ml.

34. The composition according to any one of claims 2 to 33, wherein the groundECM and the hydrogel or precursor thereof are mixed with each other.

35. The composition according to any one of claims 2 to 34, further comprising cellculture media.

36. The composition according to any one of claims 2 to 35, wherein the cell culturemedia is Dulbecco’s modified eagle’s medium (DMEM).

37. A kit comprising the ECM according to claim 1 and / or the compositionaccording to any one of claims 2 to 36, phosphate-buffered saline, cell culture media, and instructions for use.

38. The kit according to claim 37, further comprising thrombin.

39. The kit according to any one of claims 37 to 38, further comprising cells.P7250PC006840. A method of manufacturing the composition according to any one of the claims2 to 36, the method comprising the following steps: a. culturing a stem cell line, such as a mesenchymal stem cell line, in vitro;b. exposing the stem cell line to differentiation factors, such aschondrogenic factors, for at least 1 week, such as 2 weeks, such as 3 weeks, such as 4 weeks, such as 5 weeks, such as 6 weeks, such as 7 weeks, such as 8 weeks, preferably 3 weeks, thereby differentiating said stem cell line into mesenchymal lineage cell types, such as bone cells, fat cells, cartilage cells and / or muscle cells. c. culturing the mesenchymal lineage cell types, thereby producing anextracellular matrix (ECM); d. lyophilizing the ECM of step c;e. grinding the ECM of step d, thereby obtaining a ground ECM;and / or f. mixing the ground ECM with a hydrogel or precursor thereof.

41. The method according to claim 40, wherein the stem cell line is cultured on 2Dculture plates.

42. The method according to any one of claims 40 to 41, wherein the stem cell lineis cultured on 3D porous scaffolds.

43. The method according to any one of claims 40 to 42, wherein the 3D porousscaffolds are collagen scaffolds, such as sponges.

44. The method according to any one of claims 40 to 43, wherein the collagen iscollagen type I.

45. The method according to any one of claims 40 to 44, wherein the humanmesenchymal stem cell line is as a stem cell line overexpressing Bone Morphogenetic Protein-2 (BMP-2), such as the Mesenchymal Sword ofDamocles – BMP2 (MSOD-B) cell line.

46. The method according to any one of claims 40 to 45, whereinP7250PC0069 -the chondrogenic factors are selected from the group consisting oftransferrin, selenium, ascorbic acid, dexamethasone,TGF-β3, TGF-β1, BMP-2, BMP-7, and BMP-4, -the ascorbic acid concentration is at least 0.01 mM, such as 0.05 mM, suchas 0.1 mM, such as 0.2 mM, such as 0.5 mM. -the dexamethasone concentration is at least 0.01 µM, such as 0.05 µM,such as 0.1 µM, such as 0.5 µM, such as 1 µM, such as 1.5 µM, such as 2µM. -the TGF-β3 concentration is at least 1 ng / ml, such as 2 ng / ml, such as 5ng / ml, such as 10 ng / ml, such as 15 ng / ml, such as 20 ng / ml.

47. The method according to any one of claims 40 to 46, wherein the stem cell lineis exposed to differentiation factors for at least 3 days, such as at least 4 days, such as at least 5 days, such as at least 6 days, such as at least 7 days, suchas at least 8 days, such as at least 9 days, such as at least 10 days, such as atleast 11 days, such as at least 12 days, such as at least 13 days, such as atleast 14 days, such as at least 15 days, such as at least 16 days, such as atleast 17 days, such as at least 18 days, such as at least 19 days, such as atleast 20 days, such as at least 21 days, such as at least 22 days, such as atleast 23 days, such as at least 24 days, such as at least 25 days, such as atleast 26 days, such as at least 27 days, such as at least 28 days, such as atleast 29 days, such as at least 30 days, such as at least 31 days, such as atleast 32 days, such as at least 33 days, such as at least 34 days, such as atleast 35 days.

48. The method according to any one of claims 40 to 47, wherein the cells arefurther exposed to penicillin-streptomycin-glutamine, sodium pyruvate, and insulin.

49. The method according to any one of claims 40 to 48, wherein the mesenchymallineage cell types are selected from the group consisting of bone cells, fat cells, cartilage cells and / or muscle cells.

50. The method according to any one of claims 40 to 49, wherein the cells arerinsed with PBS before lyophilization.P7250PC007051. The method according to any one of claims 40 to 50, wherein the cells are snapfrozen for five minutes in liquid nitrogen before lyophilization.

52. The method according to any one of claims 40 to 51, wherein the cartilagetissue is lyophilized overnight.

53. The method according to any one of claims 40 to 52, wherein the cartilagetissue is lyophilized at -80°C.

54. The method according to any one of claims 40 to 53, wherein the cartilagetissue is lyophilized with a pressure of 0.05 mbar.

55. The method according to any one of claims 40 to 54, wherein the ECM isground with the CryoMill.

56. The method according to any one of claims 40 to 55, wherein the ECM isground in a grinding jar.

57. The method according to any one of claims 40 to 56, wherein the grinding jarhas a volume of 10 ml.

58. The method according to any one of claims 40 to 57, wherein the grinding jar isof stainless steel.

59. The method according to any one of claims 40 to 58, wherein the grinding jarcomprises two 10 mm grinding balls.

60. The method according to any one of claims 40 to 59, wherein the ECM isground for 4 x 2 minutes.

61. The method according to any one of claims 40 to 60, wherein the ECM isground at 25 hz.

62. The method according to any one of claims 40 to 61, wherein the ground ECMhas a particle size ranging from 1 to 1000 µm, such as ranging from 1 to 100 µm, such as ranging from 1 to 200 µm, ranging from 1 to 300 µm, ranging from 1 to 400 µm, ranging from 1 to 500 µm, ranging from 1 to 600 µm, ranging from 1 to 700 µm, ranging from 1 to 800 µm, ranging from 1 to 900 µm, such as ranging from 900 to 1000 µm, such as ranging from 800 to 1000 µm, such asP7250PC0071 ranging from 700 to 1000 µm, such as ranging from 600 to 1000 µm, such as ranging from 500 to 1000 µm, such as ranging from 400 to 1000 µm, such as ranging from 300 to 1000 µm, such as ranging from 200 to 1000 µm, such as ranging from 100 to 1000 µm.

63. The method according to any one of claims 40 to 62, wherein the hydrogel orprecursor thereof is synthetic and / or naturally occurring.

64. The method according to any one of claims 40 to 63, wherein the naturallyoccurring hydrogel or precursor thereof comprises fibrinogen and / or thrombin.

65. The method according to any one of claims 40 to 64, wherein the naturallyoccurring hydrogel or precursor thereof comprises alginate.

66. The method according to any one of claims 40 to 65, wherein the synthetichydrogel or precursor thereof comprises 8 to 40 % polyethylene glycols.

67. The method according to any one claims 40 to 66, wherein the hydrogel orprecursor thereof comprises at least 5 mg / ml fibrinogen, such a 6 mg / ml, suchas 7 mg / ml, such as 8 mg / ml, such as 9 mg / ml, such as 10 mg / ml, such as 11mg / ml, such as 12 mg / ml, such as 13 mg / ml, such as 14 mg / ml, such as 15 mg / ml, such as 16 mg / ml, such as 17 mg / ml, such as 18 mg / ml, such as 19mg / ml, such as 20 mg / ml.

68. The method according to any one of claims 40 to 67, wherein the hydrogel orprecursor thereof comprises 7 to 15 mg / ml fibrinogen.

69. The method according to any one of claims 40 to 68, wherein the hydrogel orprecursor thereof comprises at least 0.5 U / ml thrombin, such as 0.6 U / ml, suchas 0.7 U / ml, such as 0.8 U / ml, such as 0.9 U / ml, such as 1 U / ml, such as 1.1 U / ml, such as 1.2 U / ml, such as 1.3 U / ml, such as 1.4 U / ml, such as 1.5 U / ml, such as 1.6, U / ml, such as 1.7, U / ml, such as 1.8, U / ml, such as 1.9, U / ml, such as 2 U / ml, such as 2.1 U / ml, such as 2.2 U / ml, such as 2.3 U / ml, such as 2.4 U / ml, such as 2.5 U / ml, such as 2.6, U / ml, such as 2.7, U / ml, such as 2.8, U / ml, such as 2.9, U / ml, such as 3 U / ml.P7250PC007270. The method according to any one of claims 40 to 69, wherein the hydrogel orprecursor thereof comprises 1 to 1.5 U / ml thrombin.

71. The method according to any one of claims 40 to 70, wherein the compositioncomprises at least 0.1 mg ground ECM, such as 0.2 mg, such as 0.3 mg, such as 0.4 mg, such as 0.5 mg, such as 0.5 mg, such as 0.6 mg, such as 0.7 mg, such as 0.8 mg, such as 0.9 mg, such as 1 mg, such as 2 mg, such as 3 mg, such as 4 mg, such as 5 mg , such as 6 mg, such as 7 mg, such as 8 mg, such as 9 mg, such as 10 mg.

72. The method according to any one of claims 40 to 71, wherein the compositioncomprises 1 mg ground ECM.

73. The method according to any one of claims 40 to 72, wherein the compositionfurther comprises at least 2 µg / ml glycosaminoglycans, such as 3 µg / ml, suchas 4 µg / ml, such as 5 µg / ml, such as 6 µg / ml, such as 7 µg / ml, such as 8 µg / ml,such as 9 µg / ml, such as 10 µg / ml, such as 11 µg / ml, such as 12 µg / ml, suchas 13 µg / ml, such as 14 µg / ml, such as 15 µg / ml, such as 16 µg / ml, such as 17µg / ml, such as 18 µg / ml, such as 19 µg / ml, such as 20 µg / ml.

74. The method according to any one of claims 40 to 73, wherein the compositionfurther comprises at least 10 µg / ml Bone Morphogenetic Protein-2 (BMP-2),such as 15 µg / ml, such as 20 µg / ml, such as 25 µg / ml, such as 30 µg / ml, suchas 35 µg / ml, such as 40 µg / ml such as 45 µg / ml, such as 50 µg / ml, such as 55µg / ml, such as 60 µg / ml.

75. The method according to any one of claims 40 to 74, further comprising cellculture media.

76. The method according to any one of claims 40 to 75, wherein the cell culturemedia is Dulbecco’s modified eagle’s medium (DMEM).

77. An in vivo method for bone formation, the method comprising the followingsteps: a. administering the composition according to any one of claims 2 to 36 toa subject; andP7250PC0073 b. maintaining the composition in the subject for at least 1 week, such as 2weeks, such as 3 weeks.

78. An in vivo method for bone and bone marrow tissue formation, the methodcomprising the following steps: a. administering the composition according to any one of claims 2 to 36 toa subject; and b. maintaining the composition in the subject for at least 2 weeks, such as3 weeks, 4 weeks, such as 5 weeks, such as 6 weeks, such as 7 weeks, such as 8 weeks.

79. The method according to any one of claims 77 to 78, wherein the subject is amammal.

80. The method according to any one of claims 77 to 79, wherein the mammal is amouse, rat, rabbit, pig, dog, guinea pig or monkey.

81. The method according to any one of claims 77 to 80, wherein the administrationin vivo is a subcutaneous injection.

82. The method according to any one of claims 77 to 81, wherein the administrationin vivo is an implantation.

83. The method according to any one of claims 77 to 82, wherein at least 1 mm3,such as 2 mm3, such as 3 mm3, such as 4 mm3, such as 5 mm3, such as 6 mm3, such as 7 mm3, such as 8 mm3, such as 9 mm3, such as 10 mm3, such as 11 mm3, such as 12 mm3, such as 13 mm3, such as 14 mm3, such as 15 mm3, such as 16 mm3, such as 17 mm3, such as 18 mm3, such as 19 mm3, such as 20 mm3bone tissue is formed,84. The method according to any one of claims 77 to 83, wherein bone tissue isformed at least 1 week, such as 2 weeks, such as 3 weeks, such as 4 weeks, such as 5 weeks, such as 6 weeks post-in vivo administration.P7250PC007485. The method according to any one of claims 77 to 84, wherein the amount ofbone tissue formation is assessed by microtomography analysis.

86. The method according to any one of claims 77 to 85, further comprisingadministering cells, wherein the cells are human mesenchymal stem cells (hMSCs), genetically engineered cell lines, human hematopoieticstem / progenitor cells (HSPCs), human cancer stem cells, and / or humanimmune cells.

87. The method according to any one of claims 77 to 86, wherein the cells are stemcells, preferably mesenchymal stem cells.

88. The method according to any one of claims 77 to 87, wherein the humanmesenchymal stem cells (hMSCs) are bone marrow-derived MSCs, adipose-derived MSCs (ASCs), and / or MSCs derived from induced pluripotent stemcells (hiPSC-MSCs).

89. The method according to any one of claims 77 to 88, wherein the cells arecancer cells, such as leukemia cells, preferably acute myeloid leukemia (AML) cells.

90. The method according to any one of claims 77 to 89, wherein the human cellsare human cancer cells.

91. The method according to any one of claims 77 to 90, wherein the cancer cellsare leukemia cells.

92. The method according to any one of claims 77 to 91, wherein the cancer cellsare solid cancer cells.

93. The method according to any one of claims 77 to 92, wherein the leukemia cellsare selected from the group consisting of acute myeloid leukemia (AML) cells, acute lymphocytic leukemia (ALL) cells, chronic lymphocytic leukemia (CLL)cells, and chronic myeloid leukemia (CML) cells.P7250PC007594. The method according to any one of claims 77 to 93, wherein the solid cancercells are selected from the group consisting of bone cancer cells, prostate cancer cells, breast cancer cells, and neuroblastoma cells.

95. The method according to any one of claims 77 to 94, wherein the leukemia cellsare AML cells.

96. The method according to any one of claims 77 to 95, wherein the human cancerstem cells are multiple myeloma, osteosarcoma, metastatic breast cancer, metastatic prostate cancer, or leukemia cells.

97. The method according to any one of claims 77 to 96, wherein the formation ofbone and bone marrow is identified using radiographic imaging.

98. The method according to any one of claims 77 to 97, wherein the formation ofbone and bone marrow is identified using micro-computed tomography (µCT).

99. The method according to any one of claims 77 to 98, wherein the formation ofbone and bone marrow is identified using Safranin-O / Fast Green staining.

100. The method according to any one of claims 77 to 99, wherein theformation of bone and bone marrow is identified using Masson's Trichrome staining.

101. The method according to any one of claims 77 to 100, wherein humancells are present in the formed bone marrow microenvironment, at least 1 week,such as at least 2 weeks, such as at least 3 weeks, such as at least 4 weeks, such as at least 5 weeks, such as at least 6 weeks post-in vivo implantation.

102. A product obtained by the method according to any one of claims 77 to101.

103. A method for testing a candidate pharmaceutical product, the methodcomprising the following steps: a. establishing bone and bone marrow according to any one of claims 77to 101;P7250PC0076 b. administering pathological cells, wherein the pathological cells arepatient-derived pathological cells or genetically engineered pathologicalcells, such as cancer cells, to the bone and bone marrow of step a,thereby establishing a model pathological bone and bone marrow, such as a cancer state bone and bone marrow; c. assessing the viability and / or cell number of the pathological cells;d. administering a candidate pharmaceutical product;e. assessing the viability and / or cell number of the pathological cells;and f. classifying the candidate pharmaceutical product as effective againstpathological cells if the viability and / or cell number is decreased in step e compared to step c.

104. The method according to claim 103, wherein the patient-derivedpathological cells are primary cancer cells and their premalignant state cells,hematopoietic cells from patients with genetic disorders, marrow failure, or clonal hematopoiesis, tumor-initiating cells or cancer stem cells; and / or inflammatory or immune cells from autoimmune bone pathologies.

105. The method according to claim 103, wherein the genetically engineeredpathological cells are mesenchymal cells, hematopoietic cells, epithelial orcarcinoma-derived cells, pluripotent stem cells differentiated into bone, marrow,or cancer-relevant lineages, immortalized cell lines or patient-derived xenograft (PDX) derivatives.

106. The method according to claim 103, wherein the mesenchymal cells areMSCs and / or stromal cells.

107. The method according to claim 103, wherein the hematopoietic cells areHSCs, progenitors, and / or lineage-committed immune cells.

108. The method according to claim 103, wherein the epithelial or carcinoma-derived cells are breast, prostate, or lung cancer cells with bone metastaticpotential.P7250PC0077109. The method according to claim 103, wherein the pluripotent stem cellsdifferentiated into bone, marrow, or cancer-relevant lineages are hiPSCs.

110. The method according to claim 103, wherein the candidatepharmaceutical product is a pharmaceutical product against cancer cells.

111. The method according to any one of claims 103 to 110, wherein thepharmaceutical product is a drug.

112. The method according to any one of claims 103 to 111, wherein thepharmaceutical product is an agent used in cell therapy or gene therapy.

113. The method according to claim 103, wherein the viability and / or cellnumber of the pathological cells is assessed using hemocytometry, flow cytometry, confocal microscopy, histopathological analysis, such asimmunohistochemistry, and / or transcriptomic profiling, such as RNA sequencing.

114. The method according to claim 103, wherein the viability and / or cellnumber of the pathological cells is assessed using the markers CD34, CD117, CD33, CD45RA, and / or CD123.

115. The method according to claim 103, wherein the presence of ≥1%pathological cells is indicative of a pathological bone and bone marrow.

116. A method for identifying treatment responders, the method comprising thefollowing steps: a. obtaining cells from a patient, wherein the cells are primary cancer cellsand their premalignant state cells, hematopoietic cells from patients with genetic disorders, marrow failure, or clonal hematopoiesis, tumor- initiating cells or cancer stem cells, and / or inflammatory or immune cells from autoimmune bone pathologies; b. mixing the cells of step a. with the composition according to any one ofclaims 2 to 36; c. administering the mixture of step b. to a subject;P7250PC0078 d. maintaining the mixture in the subject for at least 2 weeks, such as 3weeks, 4 weeks, such as 5 weeks, such as 6 weeks, such as 7 weeks, such as 8 weeks, thereby establishing a model of the bone and bone marrow, of said patient, such as a cancer state bone and bone marrow; e. assessing the viability and / or cell number of the administered cells in thebone marrow; f. administering a pharmaceutical product, such as an anti-cancer drug;g. assessing the viability and / or cell number of the administered cells;and h. classifying the patient as a responder to the pharmaceutical product ifthe viability and / or cell number is decreased in step g compared to stepe.

117. The method according to claim 116, wherein the cells are cancer cells.

118. The method according to any one of claims 116 to 117, wherein thecancer cells are multiple myeloma, leukemia, metastatic solid tumors.

119. The method according to any one of claims 116 to 118, wherein thecancer cells are engrafted leukemia cells and / or engrafted human solid cancer cells.

120. The method according to any one of claims 116 to 119, wherein theleukemia cells are selected from the group consisting of acute myeloid leukemia (AML) cells, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML).

121. The method according to any one of claims 116 to 120, wherein the solidcancer cells are selected from the group consisting of bone cancer cells, prostate cancer cells, breast cancer cells, and neuroblastoma cells.

122. The method according to any one of claims 116 to 121, wherein theleukemia cells are AML cells.P7250PC0079123. The method according to any one of claims 116 to 122, wherein thesubject is a mammal.

124. The method according to any one of claims 116 to 123, wherein themammal is a mouse, rat, rabbit, pig, dog, guinea pig or monkey.

125. The composition according to any one of claims 2 to 36, for use inmedicine.

126. The composition according to any one of claims 2 to 36 for use in themethod according to any one of claims 77 to 125.

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