WO2017003877A1 - Modified extracellular matrix for enhanced stem cell homing and engraftment - Google Patents

Abstract

Disclosed are compositions comprising a decellularized ECM (extracellular matrix) and at least one signaling marker, wherein the signaling marker is coupled to the decellularized ECM. The compositions may further comprise demineralized bone matrix. In some embodiments, the decellularized ECM and the at least one signaling molecule are biotinylated. In some aspects, the at least one signaling marker is coupled to the decellularized ECM by streptavidin. Also disclosed are the methods of producing the compositions and the methods of using the compositions.

Description

MODIFIED EXTRACELLULAR MATRIX FOR ENHANCED STEM CELL HOMING

AND ENGRAFTMENT

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No.

62/187,039, filed June 30, 2015, and incorporates the disclosure of the provisional application by reference thereto.

TECHNICAL FIELD

This disclosure relates to modifications of the extracellular matrix (ECM) for enhanced homing and engraftment to sites of injury and to stem cells.

BACKGROUND

The pluripotency of stem cells, in particular mesenchymal stem cells (MSCs) hold great therapeutic potential in tissue engineering. A major limitation of existing stem cell therapies, however, is low adhesion and engraftment to sites of injury. Accordingly, methods and compositions for improved stem cell adhesion and engraftment to sites of injury is needed.

SUMMARY OF THE INVENTION

Provided are compositions comprising a decellularized ECM (extracellular matrix) and at least one signaling marker, wherein the signaling marker is coupled to the decellularized ECM. In certain embodiments, the composition further comprises demineralized bone matrix. In a particular embodiment, the decellularized ECM and the at least one signaling molecule are biotinylated. In some aspects, the at least one signaling marker is coupled to the decellularized ECM by streptavidin. Other bioconjugation technologies are available that allow for covalent attachment of chemical modifications to proteins. For example, aqueous cross-linking reagents that use "click" chemistry (a.k.a. Diels-Alder reactions), but involve modifying the target material first, preferably a synthetic polymer. However, this option is not available when dealing with "native" ECM (Nimmo 2011). In a particular embodiment, the at least one signaling marker is selected from a homing marker for the site of injury, a modifier of MSC gene expression, or both. In a non-limiting embodiment, the at least one signaling marker is a glycan. The at least one signaling marker may also be a growth factor selected from β-catenin family of proteins, BMP family of proteins, FGF family of proteins, PDGF family of proteins, and TGF superfamily of proteins. In certain specific embodiments, the growth factor from the β-catenin family of proteins is Wnt. Of the TGF superfamily, TGF-alpha is known to be a mitogenic and chemotactic factor for keratinocytes and fibroblasts. TGF-betal and TGF-beta2 promote angiogenesis and promote chemoattraction of inflammatory cells. Thus in some embodiments, the growth factor from the TGF family are selected from TGF-alpha, TGF-betal, and TGF- betal. In the BMP family proteins, BMP1, BMP5, and BMP8a are involved in cartilage development while BMP2 and BMP7 are important for bone development (Gabriel et al. 2009). Accordingly, the growth factor from the BMP family of proteins is selected from BMP-1, BMP- 2, BMP5, BMP7, and BMP8a.

In some embodiments, the composition further comprises a dressing or a bandage, wherein the decellularized ECM is applied to the dressing or bandage. Examples of the dressing or bandage include gauze, adhesive bandage, or liquid bandage. In these embodiments, the composition may further comprise an antimicrobial agent.

The decellularized ECM maybe isolated from a MSC culture. For example, the decellularized ECM is isolated from a fibroblast culture. In some aspects, the fibroblast culture comprises fibroblasts of at least one tissue origin selected from the group consisting of neural, epidermal, dermal, adipose, cardiac, kidney, muscle, liver, cartilage, pancreas, endometrium of uterus, umbilical cord, dental pulp, trabecular bone, and cortical bone. The decellularized ECM may also be isolated from a stromal cell culture, for example, a stromal cell culture comprising marrow stromal cells.

Also provided herein are methods of targeting tissue engineering treatment to a site of injury comprising administering the composition of the invention, wherein the signaling marker is a homing marker for the site of injury; and administering to the subject MSCs. In some implementations, the composition of the invention is administered systemically. In other implementations, administering the composition comprises applying a composition comprising the modified decellularized ECM and/or demineralized bone matrix to the site of injury. In some implementations the MSC is administered systemically, while in other implementations, the MSC is administered locally. Methods of improving the therapeutic differentiation of MSCs in a subject are also provided. Such methods comprise use of the composition of the invention, wherein the signaling marker is modifier of MSC gene expression; and administering to the subject MSCs. In some aspects, the modifier of MSC gene expression may be a growth factor selected from β-catenin family of proteins, BMP family of proteins, FGF family of proteins, PDGF family of proteins, and TGF superfamily of proteins. In certain specific embodiments, the growth factor from the β- catenin family of proteins is Wnt. Of the TGF superfamily, TGF-alpha is known to be a mitogenic and chemotactic factor for keratinocytes and fibroblasts. TGF-betal and TGF-beta2 promote angiogenesis and promote chemoattraction of inflammatory cells. Thus in some embodiments, the growth factor from the TGF family are selected from TGF-alpha, TGF-betal, and TGF-beta2. In the BMP family proteins, BMPl, BMP 5, and BMP 8 a are involved in cartilage development while BMP2 and BMP7 are important for bone development (Gabriel et al. 2009). Accordingly, the growth factor from the BMP family of proteins is selected from BMP-1, BMP- 2, BMP5, BMP7, and BMP8a.

In some aspects, the composition of the invention administered for improving the therapeutic differentiation of MSCs in a subject further comprises a signaling marker that is a homing marker for the site of injury. In some implementations, the composition of the invention is administered systemically. In other implementations, administering the composition of the invention comprises applying a composition comprising the modified decellularized ECM and demineralized bone matrix to the site of injury. In some implementations the MSC is administered systemically, while in other implementations, the MSC is administered locally.

The invention also provides methods of modifying ECM comprising producing ECM from cultured cells; isolating ECM produced by cultured cells; decellularizing the isolated ECM; biotinylating the ECM; biotinylating a signaling marker; and combining the biotinylated ECM and the biotinylated signaling markers with streptavidin.

In another embodiment, the invention provides a tumor cell-specific antibody composition. In some aspects, the composition is specific to the tumor cells of a specific subject. The tumor cell-specific antibody composition comprises a fluorescent label and an anti-cancer antibody produced using a culture of a tumor cells, where the fluorescent label is coupled to the anti-cancer antibody. The culture of tumor cells is produced by culturing, and thus expanding, tumor cells isolated from a subject on the above-described composition comprising decellularized ECM and a signaling molecule. The anti-cancer antibody is produced by administering the expanded tumor cells to a host to induce an immune response in the host and harvesting serum from the host to extract antibodies against the expanded tumor cells. In some implementations, the tumor cell-specific antibody composition further comprises a compound that selectively targets and kills the tumors cells isolated from the subject. In some embodiments, the compound is identified by screening anti-cancer agents using a culture of tumor cells produced by culturing, and thus expanding, tumor cells isolated from a subject on the above- described composition comprising decellularized ECM and a signaling molecule. Where the anticancer agent increases cell death in the culture of expanded tumor cells, the anti-cancer agent kills the tumors cells isolated from the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a schematic of how the binding of ECM to cell-surface integrin pairs can evoke integrin-dependent translational control such as de-repression of global protein synthesis.

Figure 2 depicts the chemical modification of ECM by coupling to biotin-streptavidin glycan ligand.

Figure 3 depicts targeting of systemically introduced homing ECM to a site of injury wherein the homing ECM binds to lectins that present from the surface of epithelial cells in response to inflammatory cytokines released at sites of injury.

Figure 4 is a histogram summarizing the results of an antibody microarray analysis of soluble secreted ECM from adipose-derived stem cells (ADSCs).

Figure 5 is a heat map showing semi-quantitative values for expressed cytokines and growth factors in the concentrated stem cell conditioned media.

DETAILED DESCRIPTION

As used herein, the verb "comprise" as is used in this description and in the claims and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there is one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one." The present invention relates to the discovery that ECM can be enriched for protein ligands beneficial to the retention, viability, proliferation, and differentiation of surface-attached MSCs. Suitable ECM includes, for example, ECM secreted by tissue culture cells, including cultured stem cells, or artificially created ECM.

In tissue culture, the issue of low MSC adhesion is addressed through using components of cell-secreted ECM as an attachment medium. In order for stem cells to adhere to tissue culture plastic, they will secrete specialized glycoproteins that provide structure and an attachment substrate for cells. Molecules extruded into stem cell-secreted ECM include those that can act as signals to attract cells (homing), ensure cell viability ("waking up" otherwise quiescent progenitor cells), and encourage cell proliferation, specify differentiation, and promote vascularization. It is the role of the ECM, therefore, to not only provide a structural foundation for building tissues, but to also act as a reservoir of signaling molecules that can guide cells to repair injured tissues. Proteinaceous molecules in the ECM serve as binding ligands for cell- surface molecules known as integrins. By engaging binding with certain integrins on the cell surface of stem cells recruited from the circulation, certain stimulatory signal -transduction pathways can be activated. Examples of these compounds (see Figure 1 ) include the variant protein domains of fibronectin: the V-region domain (binds integrin ανβ3), the III- 10 domain (binds integrin α2β 1), and full-length fibronectin (binds integrin α5β1); vitronectin (binds integrin ανβ3), laminin (binds integrin αόβ4), and fibrinogen (binds integrin αΙ¾β3). Similarly, blocking antibodies to specific integrins have been shown to shut-off activation of other undesirable signal-transduction cascades,

Proof-of-concept studies have already demonstrated some of the desirable properties of ECM that makes it amenable to engineering for biomedical applications, such as infusing it into tissue allograft materials for in vivo applications. It has been demonstrated that homogenized and transferred decellularized ECM exhibits a potent ability to enhance cellular differentiation comparable to its performance in the native state (Decaris et al. 2012). In addition, alteration of in vitro environmental culture conditions during stem cell ECM production affects the capacity of that decellularized ECM to modulate cell fate (Decaris and Leach 2010). The ECM from bone marrow stem cells and/or stromal cells has also been shown to promote cell viability, which is an attractive property for an application in regenerative medicine and surgical repair of tissues. Specifically, stem cell ECM can extend the reproductive capacity of undifferentiated progenitor cells to higher passage numbers in vitro (Chen 2010). Even more remarkable is the ability of stem cell ECM to rescue older cells and impart them with more regenerative potential (Sun et al. 2011).

In one embodiment, the present invention is directed to a composition comprising decellularized ECM and at least one signaling marker. In some aspects, the ECM may be stem cell-secreted. In some embodiments, the decellularized ECM is modified, for example chemically, with the at least one signaling marker. Chemical modification of the decellularized ECM required to modify stem cell-secreted ECM to enhance homing are straightforward and widely used biochemical techniques. Chemical coupling using the biotin/streptavidin protein- ligand system conjugated to glycan moieties (d) has been shown to enhance homing of MSCs to inflamed lesions in vivo (Figure 3) (Sarkar et al. 2011). The biotin-strepatavidin system is most often used in biological applications for several reasons. Firstly, biotin-streptavidin tends to be highly biocompatible (biotin is also known as Vitamin B7). Secondly, these tags tend to not interfere with the function of their tagged proteins. Furthermore, engineered variants of biotin and streptavidin have been designed to alleviate issues such as aggregation (monomeric versus tetrameric streptavidin) (Dundras 2014) and stearic hindrance (varying spacer arm lengths attached to biotin). Also streptavidin is used extensively in molecular biology and biotechnology due to the streptavidin-biotin complex's resistance to organic solvents, denaturants, detergents, proteolytic enzymes, and temperature and pH extremes. Biotin tends to use Sulfo- HS chemistry to covalently bind to the N-terminus or primary amine group (-NH₂) of peptides, or the side groups of lysine (also -NH₂). However, alternative chemistries that allow for crosslinking to different functional groups such as oxidized sugar and carbohydrates, C-terminal peptide groups or aspartate and glutamate side chains (i.e. carboxylic acid), and sulfhydryl groups (reduced free thiols on cysteine residues).

Application of this chemical modification technique to the myriad of proteins present in stem cell-secreted ECM can be used to create a "homing ECM" that would localize to sites of inflammation and injury in vivo. In some embodiments, the decellularized ECM may be chemically modified with at least one signaling marker at the reactive groups on lysine residues using amine-reactive succinimidyl esters. Amino acid residues other than lysine may be targeted for bioconjugation, for example, cysteine, tyrosine, tryptophan, or the residues at the C- and/or N-terminus of proteins. In some implementations, the decellularized ECM may be modified through a combination of chemical modification methods. In order to crosslink different compounds to proteins present in the ECM, a variety of different bioconjugation chemistries can be deployed. These include carboxyl-to-amine reactive groups (e.g. carbodiimide), amine- reactive groups (e.g. NHS ester, imidoester), sulfhydryl-reactive groups (e.g. maleimide, haloacetyl, pyridyldisulfide), aldehyde-reactive groups (e.g. hydrazide, alkoxyamine), photoreactive groups (e.g. diazirine, aryl azide), and hydroxyl (nonaqueous)-reactive groups (e.g. isocyanate). Preferentially, amine-reactive chemistry would be used since the targeted reactive group (primary amines) on the protein to be labeled is more accessible for conjugation because of its positive charge under physiological conditions. This strategy also selects for conjugation that is least likely to denature the protein structure, and thus less likely to alter the function of that protein (Thermo Scientific Crosslinking Technical Handbook: Easy molecular bonding crosslinking technology 2012).

The signaling marker may be specific protein-binding ligands. The signaling marker may enhance homing of the cells to the decellularized ECM (e.g. MSC homing marker) and/or enhance homing of the ECM to a site of injury (homing marker for the site of injury). The signaling marker may be a glycan, a growth factor, or a cytokine. The glycan may be a glycan containing Sialyl-Lewis^x. These glycans are recognized by E-Selectin and P-Selectin which are expressed at the site of injury (Sarkar et al. 2011). The glycan may also be a VCAM (vascular cell adhesion molecule) or an ICAM (intercellular adhesion molecule). The growth factor may be selected from the group consisting of β-catenin family of proteins (e.g. Wnts), BMP family of proteins (BMPs, for example, BMP-1, BMP-2, BMP5, BMP7, and BMP 8 a), FGF family of proteins (FGFs), insulin-like growth factors (IGFs), PDGF family of proteins (PDGFs), and TGF superfamily of proteins (TGFs, for example, TGF-alpha, TGF-betal, and TGF-beta2). In some embodiments, the growth factor may be selected from the group consisting of vascular endothelial growth factor (VEGF), hypoxia inducible factor- la (HIF-la), plasminogen activator inhibitor (e.g. PAI-1), neuregulin-1 (NRG1), and hepatocyte growth factor (HGF), The cytokine may be a chemokine, for example, a stromal-derived factor or related proteins. In some aspects, the cytokine may SDF-Ια (also known as CXCL12). The signaling marker may also be osteopontin (OPN).

In some aspects, the signaling marker may be a recombinant purified protein known to mediate engagement of surface adhesion proteins and influence differentiation of progenitor cells (for example, MSCs). When such signaling marker is added to the decellularized ECM, the instructive potential of the ECM is augmented. Additional proteins would include full-length fibronectin and its splice variants, notably the human orthologues of the rat fibronectin EIIIA, EIIIB, III- 10 and V-region domains (Fernandez et al. 2010). Similarly, adhesion proteins (and their variants) identified as parts of the "basement membrane toolkit" that bind cell-surface integrins (e.g. laminin, vitronectin, biglycan, tenascin, etc.) or tether growth factors (e.g. heparin, heparan sulfate proteoglycans, etc.) could be added or chemically coupled to the decellularized ECM (Hynes 2009). The signaling marker may also be selected from the group consisting of collagen (e.g. types I, II, III, IV, and V), laminin, glucosaminoglycan (GAG), carcinoembiyonic antigen-related cell adhesion molecule 1 (CEA-CAM1), versican, periostin, developmental endothelial locus- 1 (del- 1 ), and nephronectin and the related EGFL-1.

The signaling marker may also be a biomolecule that triggers gene expression that is typically activated in response to binding of cell-surface integrins to specific ligands (Figure 3). These solid-phase ligands that are incorporated into the ECM can potentially wake up otherwise quiescent stem cells and stimulate the global protein synthesis machinery (Chung and Kim 2008). Similarly, stimulation of a subset of gene products can also be enabled through binding of cell-surface integrins to ECM components, i.e., binding through integrin α6β4 facilitates both gene transcription (Soung et al. 2011) and translation initiation factor eIF4E-dependent protein expression of VEGF, a secreted growth factor known to drive angiogenesis and vascularization (Korneeva et al. 2010). A signaling marker that triggers gene expression changes may also be selected from the group consisting of epidermal growth factor receptor (EGFR), platelet derived growth factor receptor (PDGFR), epidermal growth factor (EGF), and platelet derived growth factor (PDGF).

In some implementations, the signaling marker may be the mechanism by which decellularized ECM is visible to imagining technologies such as fluorescence, luminescence, or ultrasound. The use of a catheter to deliver stem cells (Dib et al 2002) or ECM to sites of cardiac tissue injury (Singelyn et al. 2012) has been demonstrated in studies that sought to heal cardiac tissue. Next-generation catheter technologies that utilize ultrasound for needle-tip guidance would be able to image delivered biomaterials such as the decellularized ECM, or stem cells microencapsulated in the modified decellularized ECM (Mayfield et al. 2014) provided the modified decellularized ECM was rendered radiopaque or sonically opaque ("echogenic"). Accordingly, the signaling marker may be contrast agents, fluorescent dyes, and luminescent molecules for visualization of delivery and localization of the biomaterial using existing imaging technologies. The signaling marker may also be a biocompatible echogenic compounds, for example, hydroxy apatite fluorapatite, iodapatite, or carbonate-apatite (Tiwari A and Tiwari A, eds. 2014). Similarly, the signaling marker may be a dye that generates fluorescent, luminescent, or colorimetric targets for imaging could also be deployed with the modified decellularized ECM (hereinafter "homECM").

In one aspect, the composition further comprises a human tissue allograft, for example, demineralized, decellularized bone spacers derived from cadaveric human tissue or dermal matrix allografts. The homECM could augment the regenerative properties of surgical tissue allografts, enhancing their healing potential, strengthening rebuilt tissue, and accelerating healing times. Although the gold standard for surgical grafting interventions is the autograft, which uses the patient's own tissue, trauma and morbidity at the donor site makes allograft transplants a valued industry alternative. Typically, tissue allografts are derived from cadaveric human tissue such as bone, dermis, and tendon, which are decellularized and processed for minimal immunological rejection by the host/recipient. These allografts are used for performing spinal fusions, breast reconstructions, and surgical repair of sports medicine injuries.

The stem cell instructional content of homECM may be further enhanced by altering the culture conditions of the ECM-producing cells. Specific environmental conditions can induce markers related to osteogenic differentiation, bone mineralization and ECM deposition. The imposed parameters include variations on culture duration, oxygen tension, cell seeding density, and media composition (Decaris and Leach 2010).

In another embodiment, the invention is directed to methods for chemically modifying the native ECM involves several manipulations comprising: 1) altering growth conditions for cultured cells to enrich secreted ECM for cell viability signals, 2) decellularizing deposited ECM to retain instructional potential and be utilized as an acellular graft, and 3) chemically modifying decellularized ECM to enhance its ability to "home in" on sites of inflammation and tissue repair. Thus in some aspects, the methods of modifying the ECM may comprise producing ECM from cultured cells; isolating ECM produced by cultured cells; decellularizing the isolated ECM; biotinylating the ECM; biotinylating a signaling marker; and combining the biotinylated ECM and the biotinylated signaling markers with streptavidin. The cultured cells may be a MSC culture, a fibroblast culture, or a stromal cell culture. In some aspects, the fibroblast culture comprises fibroblasts of at least one tissue origin selected from the group consisting of neural, epidermal, dermal, adipose, cardiac, kidney, muscle, liver, cartilage, pancreas, endometrium of uterus, umbilical cord, dental pulp, trabecular bone, and cortical bone. The stromal cell culture may comprise marrow stromal cells.

The growth conditions may be altered decreasing oxygen tension from normoxic levels (ambient oxygen) from 21% to 5% (hypoxia) (Decaris & Leach (2011) to encourage angiogenic factors, such as VEGF (Polo-Corrales et al. 2014). Growth condition alterations may also include changing the cell media compositions to favor macromolecular crowding and thus increased ECM deposition (Satyam 2014). For example, the cell media comprises 100 μg/mL dextran sulfate of MW ~500kDa, 37.5 mg/mL Ficoll 70, 25 mg/mL Ficoll 400 (Sigma-Aldritch), and 75 μg/mL carrageenan.

The invention is also directed to methods of targeting tissue engineering treatment to a site of injury and methods of improving the therapeutic differentiation of MSCs in a subject. Both methods comprise administering to the subject the modified decellularized ECM of the composition and MSCs. The decellularized ECM could be administered systematically (e.g. intravenously) to find sites of injury and deliver a substrate for stem cell recruitment. Alternatively, the decellularized ECM could be applied administered locally, for example, in a composition comprising human allograft tissue (e.g. demineralized bone matrix or decellularized demineralized bone allograft). Thus homECM would be administered locally. The engrafted homECM then provides an anchored substrate that can enhance recruitment of circulating or injected stem cells, optimize their regenerative properties, enhance cellular retention and amplify their healing potential. The complex mixture of information-rich macromolecules contained within stem cell-produced ECM has the capacity to attract circulating progenitor cells to lesions, whereupon included soluble protein signals modulate cell fate. Accordingly, the MSC may be administered locally or systemically.

For methods of targeting tissue engineering treatment to a site of injury, the signaling marker may be a homing marker for the site of injury. For methods of improving the therapeutic differentiation of MSCs in a subject, the signaling marker may be a modifier of MSC gene expression. The at least one signaling marker of the modified decellularized ECM for improving the therapeutic differentiation of MSCs in a subject may also include a homing marker for the site of injury.

The present invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference in their entirety for all purposes.

EXAMPLES

Elements and acts in the example are intended to illustrate the invention for the sake of simplicity and have not necessarily been rendered according to any particular sequence or embodiment. The example is also intended to establish possession of the invention by the Inventors. Cytokines Expressed in Soluble Secreted ECM From Adipose-Derived Stem Cells (ADSCs)

We recovered the ADSCs from cadaveric tissue, cultured them in the laboratory, and then collected the conditioned medium, which contains secreted soluble ECM. The conditioned medium was concentrated by at least 50-fold using centrifuge microconcentrators. The concentrated conditioned medium was then hybridized with RayBiotech's antibody microarray Human Cytokine Array CI 000 (product code AAH-CYT-1000), which detects 120 human cytokines. This Cytokine Array utilizes the sandwich immunoassay principle, where a panel of capture antibodies is printed on a nitrocellulose membrane solid support. The array membranes are processed similarly to a Western blot (chemiluminescent readout). Signals are then visualized on x-ray film or a digital image, allowing densitometry data collection and calculation of fold- changes for each detected protein. Densitometry data was compiled using the free software package NIH ImageJ. Relative levels of cytokines were compared between concentrated conditioned media (soluble secreted ECM) and complete media (as a control). Selected cytokines that showed notable differences in expression were used to generate the histogram to show fold- changes (Y-axis) versus identified cytokines (X-axis) (Figure 4). Table 1 lists the significant cytokines from the histogram and explains their biological activities. Table 1.

Marker Biological activity

Plays a role in weight homeostasis. Involved in the control of feeding behavior through the central melanocortin system. Acts as alpha melanocyte-stimulating hormone

Ag F

antagonist by inhibiting cAMP production mediated by stimulation of melanocortin receptors within the hypothalamus and adrenal gland. Has very low activity with MC5R (By similarity). Is an inverse agonist for MC3R and MC4R being able to suppress their protein)

constitutive activity. It promotes MC3R and MC4R endocytosis in an arrestin- dependent manner

Receptor tyrosine kinase that transduces signals from the extracellular matrix into the cytoplasm by binding growth factor GAS6 and which is thus regulating many physiological processes including cell survival, cell proliferation, migration and differentiation. Ligand binding at the cell surface induces dimerization and

autophosphorylation of AXL. Following activation by ligand, ALX binds and induces tyrosine phosphorylation of PI3-kinase subunits PIK3R1, PIK3R2 and PIK3R3; but also GRB2, PLCGl, LCK and PTPNl 1. Other downstream substrate candidates for AXL are CBL, NCK2, SOCS1 and TNS2. Recruitment of GRB2 and phosphatidylinositol 3

A.d

kinase regulatory subunits by AXL leads to the downstream activation of the AKT kinase. GAS6/AXL signaling plays a role in various processes such as endothelial cell protein kkrsss

survival during acidification by preventing apoptosis, optimal cytokine signaling during receptor

human natural killer cell development, hepatic regeneration, gonadotropin-releasing

UFO)

hormone neuron survival and migration, platelet activation, or regulation of thrombotic responses. Plays also an important role in inhibition of Toll-like receptors (TLRs)- mediated innate immune response. (Microbial infection) Acts as a receptor for lassa virus and lymphocytic choriomeningitis virus, possibly through GAS6 binding to phosphatidyl-serine at the surface of virion envelope (PubMed:22156524,

PubMed:22673088, PubMed:25277499, PubMed:21501828). Acts as a receptor for ebolavirus, possibly through GAS6 binding to phosphatidyl-serine at the surface of virion envelope (PubMed: 17005688)

nvolved in neutrophil activation. In vitro, ENA-78(8-78) and ENA-78(9-78) show a

CXCL5

threefold higher chemotactic activity for neutrophil granulocytes

CXCL2: Produced by activated monocytes and neutrophils and expressed at sites of inflammation. Hematoregulatory chemokine, which, in vitro, suppresses hematopoietic

GRO a/b/g progenitor cell proliferation. GRO-beta(5-73) shows a highly enhanced hematopoietic iCXCXl/CXC activity. CXCL3: Ligand for CXCR2 (By similarity). Has chemotactic activity for

L2/CXCL3) neutrophils. May play a role in inflammation and exert its effects on endothelial cells in an autocrine fashion. In vitro, the processed form GRO-gamma(5-73) shows a fivefold higher chemotactic activity for neutrophilic granulocytes

Has chemotactic activity for neutrophils. May play a role in inflammation and exerts its

GRO alpha effects on endothelial cells in an autocrine fashion. In vitro, the processed forms GRO- (CXCLl) alpha(4-73), GRO-alpha(5-73) and GRO-alpha(6-73) show a 30-fold higher

chemotactic activity

(lasui -like

IGF -binding proteins prolong the half-life of the IGFs and have been shown to either inhibit or stimulate the growth promoting effects of the IGFs on cell culture. They alter factor- the interaction of IGFs with their cell surface receptors

pr&ieisi 6) Marker Biological activity

The insulin-like growth factors are structurally and functionally related to insulin but have a much higher growth-promoting activity. May be a physiological regulator of [1- 14C]-2-deoxy-D-glucose (2DG) transport and glycogen synthesis in osteoblasts.

Stimulates glucose transport in bone-derived osteoblastic (PyMS) cells and is effective at much lower concentrations than insulin, not only regarding glycogen and DNA synthesis but also with regard to enhancing glucose uptake. May play a role in synapse maturation . Ca2+-dependent exocytosis of IGF 1 is required for sensory perception of smell in the olfactory bulb (By similarity). Acts as a ligand for IGFIR. Binds to the alpha subunit of IGFIR, leading to the activation of the intrinsic tyrosine kinase activity which autophosphorylates tyrosine residues in the beta subunit thus initiatiating a cascade of down-stream signaling events leading to activation of the PI3K-AKT/PKB and the Ras-MAPK pathways. Binds to integrins ITGAV:ITGB3 and ITGA6:ITGB4. Its binding to integrins and subsequent ternary complex formation with integrins and IGFR1 are essential for IGF1 signaling. Induces the phosphorylation and activation of IGFR1, MAPK3/ERK1, MAPK1/ERK2 and AKT1

L-8 is a chemotactic factor that attracts neutrophils, basophils, and T-cells, but not monocytes. It is also involved in neutrophil activation. It is released from several cell

IL-S (CXCL- types in response to an inflammatory stimulus. IL-8(6-77) has a 5-10-fold higher S activity on neutrophil activation, IL-8(5-77) has increased activity on neutrophil

activation and IL-8(7-77) has a higher affinity to receptors CXCR1 and CXCR2 as compared to IL-8(l-77), respectively

T-4

{Neurotropic Target-derived survival factor for peripheral sensory sympathetic neurons

Acts as decoy receptor for TNFSF11/RANKL and thereby neutralizes its function in

OPG

osteoclastogenesis. Inhibits the activation of osteoclasts and promotes osteoclast apoptosis in vitro. Bone homeostasis seems to depend on the local ratio between TNFSF11 and TNFRSF1 IB. May also play a role in preventing arterial calcification.

(T FRSFJ I B

May act as decoy receptor for TNFSFIO/TRAIL and protect against apoptosis.

TNFSFIO/TRAIL binding blocks the inhibition of osteoclastogenesis

Complexes with metalloproteinases (such as collagenases) and irreversibly inactivates them by binding to their catalytic zinc cofactor. Known to act on MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-13, MMP-14, MMP-15, MMP-16 and MMP-19

^'FRAIL Receptor for the cytotoxic ligand TRAIL. Lacks a cytoplasmic death domain and hence

(TN^' SF IOC is not capable of inducing apoptosis. May protect cells against TRAIL mediated

apoptosis by competing with TRAIL-Rl and R2 for binding to the ligand

uFAR

(Or ksssa e Acts as a receptor for urokinase plasminogen activator. Plays a role in localizing and lasm nogen promoting plasmin formation. Mediates the proteolysis-independent signal transduction activator activation effects of U-PA. It is subject to negative-feedback regulation by U-PA which cleaves it into an inactive form.

In response to the presence of allergens, this protein directly promotes the accumulation of eosinophils, a prominent feature of allergic inflammatory reactions. Binds to CCR3. Marker Biological activity

May play a role in the trafficking of activated/effector T-lymphocytes to inflammatory sites and other aspects of activated T-lymphocyte physiology. Chemotactic for

L·L·&^

monocytes, dendritic cells and natural killer cells. Mild chemoattractant for primary activated T-lymphocytes and a potent chemoattractant for chronically activated T- chemokine

lymphocytes but has no chemoattractant activity for neutrophils, eosinophils, and resting T-lymphocytes. Binds to CCR4. Processed forms MDC(3-69), MDC(5-69) and MDC(7-69) seem not be active.

CXCL9 (C-X- Cytokine that affects the growth, movement, or activation state of cells that participate in immune and inflammatory response. Chemotactic for activated T-cells. Binds to ch okkia 9} CXCR3

Chemotactic factor that attracts T-cells and monocytes, but not neutrophils, eosinophils, rao£sf or B-cells. Acts mainly via CC chemokine receptor CCR1. Also binds to CCR3.

chemokme CCL 15 (22-92), CCL15(25-92) and CCL 15 (29-92) are more potent chemoattractants

15) than the small-inducible cytokine A15

Growth regulator. Inhibits the proliferation of a number of tumor cell lines.

Stimulates proliferation of AIDS-KS cells. It regulates cytokine production, including

OSM IL-6, G-CSF and GM-CSF from endothelial cells. Uses both type I OSM receptor (heterodimers composed of LIPR and IL6ST) and type II OSM receptor (heterodimers )

composed of OSMR and IL6ST). Involved in the maturation of fetal hepatocytes, thereby promoting liver development and regeneration (By similarity)

A heat map (Figure 5) shows relative expressions of proteins ranging from low or background level (blue color) to high level (red color) based on the results of the Human Cytokine Array CI 000. Table 2 highlights some of the highly expressed cytokines in soluble ECM that play an important role in regulation of regeneration, inflammation and matrix remodeling. Table 3 lists the significant cytokines and growth factors from the heat map and explains their biological activities.

Table 2.

Table 3.

s an tssue n tors o meta oprotenases s . n nnate mmunty, Marker Biological activity

modulates the activity and function of neutrophils by increasing chemotaxis and the secretion of oxygen radicals. Increases phagocytosis by macrophages and enhances secretion of pro-inflammatory mediators. Increases cytotoxic ability of NK cells. Plays a pro-inflammatory role, in synergy with IL1B, by inducing NOS2 wich promotes the production of IL6, IL8 and Prostaglandin E2, through a signaling pathway that involves

JAJN- , rijjN., iviAr jN.i/iviJiJN.1 ana

in aaapuve immunity, promotes ine switch of memory T-cells towards T helper- 1 cell immune responses (By similarity). Increases CD4+CD25- T-cell proliferation and reduces autophagy during TCR (T-cell receptor) stimulation, through MTOR signaling pathway activation and BCL2 up- regulation

Cytokine that binds to TNFRSF3/LTBR. Binding to the decoy receptor TNFRSF6B

IM ^'SI 14 modulates its effects. Activates NFKB, stimulates the proliferation of T-cells, and inhibits growth of the adenocarcinoma HT-29. Acts as a receptor for Herpes simplex virus Cytokine that plays an essential role in the regulation of survival, proliferation and differentiation ot hematopoietic precursor cells, especially mononuclear phagocytes, such as macrophages and monocytes. Promotes the release of proinflammatory chemokines, and thereby plays an important role in innate immunity and in inflammatory

SI processes. Plays an important role in the regulation of osteoclast proliferation and

differentiation, the regulation of bone resorption, and is required for normal bone development. Required for normal male and female fertility. Promotes reorganization of the actin cytoskeleton, regulates formation of membrane ruffles, cell adhesion and cell iiii^i uii. -Tia s a I UIC in iipupiuicm cicai uicc

Growth factor that plays an essential role in the regulation of embryonic development, cell proliferation, cell migration, survival and chemotaxis. Potent mitogen for cells of mesenchymal origin. Required for normal proliferation and recruitment of pericytes

I XiF-Hli and vascular smooth muscle cells in the central nervous system, skin, lung, heart and placenta. Required for normal blood vessel development, and for normal development of kidney glomeruli. Plays an important role in wound healing. Signaling is modulated by the formation of heterodimers with PDGFA (By similarity)

Chemoattractant for blood monocytes, memory T-helper cells and eosinophils. Causes the release or histamine from basophils and activates eosinophils. May activate several chemokine receptors including CCR1, CCR3, CCR4 and CCR5. One of the major HIV- suppressive factors produced by CD8+ T-cells. Recombinant RANTES protein induces a dose-dependent inhibition of different strains of HrV-1, HIV-2, and simian immunodeficiency virus (SrV). The processed form RANTES(3-68) acts as a natural

R \\ ΙΊ S chemotaxis inhibitor and is a more potent inhibitor of HrV-1 -infection. The second

(ί processed form RANTES(4-68) exhibits reduced chemotactic and HIV-suppressive activity compared with RANTES(l-68) and RANTES(3-68) and is generated by an unidentified enzyme associated with monocytes and neutrophils. May also be an agonist of the G protein-coupled receptor GPR75, stimulating inositol trisphosphate production and calcium mobilization through its activation. Together with GPR75, may play a role in neuron survival through activation of a downstream signaling pathway involving the PI3, Akt and MAP kinases. By activating GPR75 may also play a role in insulin secretion by islet cells Ligand for the receptor-type protein-tyrosine kinase KIT. Plays an essential role in the regulation of cell survival and proliferation, hematopoiesis, stem cell maintenance, gametogenesis, mast cell development, migration and function, and in melanogenesis.

S( Γ

KITLG/SCF binding can activate several signaling pathways. Promotes phosphorylation of PIK3R1, the regulatory subunit of phosphatidylinositol 3-kinase, and subsequent activation of the kinase AKT1. KITLG/SCF and KIT also transmit signals via GRB2 and activation Marker Biological activity

of RAS, RAF1 and the MAP kinases MAPK1/ERK2 and/or MAPK3/ERK1. KITLG/SCF and KIT promote activation of STAT family members STAT1, STAT3 and STAT5.

KITLG/SCF and KIT promote activation of PLCG1, leading to the production of the cellular signaling molecules diacylglycerol and inositol 1,4,5-trisphosphate. KITLG/SCF acts synergistically with other cytokines, probably interleukins

Chemoattractant active on T-lymphocytes, monocytes, but not neutrophils. Activates the C-

X-C chemokine receptor CXCR4 to induce a rapid and transient rise in the level of intracellular calcium ions and chemotaxis. Also binds to atypical chemokine receptor ACKR3, which activates the beta-arrestin pathway and acts as a scavenger receptor for SDF-1. SDF-l-beta(3-72) and SDF-l-alpha(3-67) show a reduced chemotactic activity. Binding to cell surface proteoglycans seems to inhibit formation of SDF-1 -alpha(3-67) and thus to preserve activity on local sites. Acts as a positive regulator of monocyte migration snn - and a negative regulator of monocyte adhesion via the LYN kinase. Stimulates migration of alplia monocytes and T-lymphocytes through its receptors, CXCR4 and ACKR3, and decreases monocyte adherence to surfaces coated with ICAM-1, a ligand for beta-2 integrins.

SDF1A/CXCR4 signaling axis inhibits beta-2 integrin LFA-1 mediated adhesion of monocytes to ICAM-1 through LYN kinase. Inhibits CXCR4-mediated infection by T-cell line-adapted HIV-1. Plays a protective role after myocardial infarction. Induces down- regulation and internalization of ACKR3 expressed in various cells. Has several critical functions during embryonic development; required for B-cell lymphopoiesis, myelopoiesis in bone marrow and heart ventricular septum formation

Chemotactic factor for T-lymphocytes but not monocytes or granulocytes. May play a role

C ( 1.1 7 in T-cell development in thymus and in trafficking and activation of mature T-cells. Binds to CCR4

Multifunctional protein that controls proliferation, differentiation and other functions in many cell types. Many cells synthesize TGFB 1 and have specific receptors for it. It positively and negatively regulates many other growth factors. It plays an important role in bone remodeling as it is a potent stimulator of osteoblastic bone formation, causing chemotaxis, proliferation and differentiation in committed osteoblasts. Can promote either T-helper 17 cells (Thl7) or regulatory T-cells (Treg) lineage differentiation in a

I Gl ^'b- l concentration-dependent manner. At high concentrations, leads to FOXP3 -mediated

suppression of RORC and down-regulation of IL-17 expression, favoring Treg cell development. At low concentrations in concert with IL-6 and IL-21, leads to expression of the IL-17 and IL-23 receptors, favoring differentiation to Thl7 cells. Mediates SMAD2/3 activation by inducing its phosphorylation and subsequent translocation to the nucleus. Can induce epithelial-to-mesenchymal transition (EMT) and cell migration in various cell types

i cii- b- Involved in embryogenesis and cell differentiation.

Produced by activated monocytes and neutrophils and expressed at sites of inflammation.

⁽ iRO a b u Hematoregulatory chemokine, which, in vitro, suppresses hematopoietic progenitor cell proliferation. GRO-beta(5-73) shows a highly enhanced hematopoietic activity

Has chemotactic activity for neutrophils. May play a role in inflammation and exerts its effects on endothelial cells in an autocrine fashion. In vitro, the processed forms GRO-

(^"iRO alpha

alpha(4-73), GRO-alpha(5-73) and GRO-alpha(6-73) show a 30-fold higher chemotactic activity

IL-8 is a chemotactic factor that attracts neutrophils, basophils, and T-cells, but not monocytes. It is also involved in neutrophil activation. It is released from several cell types

I I . -X

in response to an inflammatory stimulus. IL-8(6-77) has a 5-10-fold higher activity on neutrophil activation, IL-8(5-77) has increased activity on neutrophil activation and IL-8(7-

pat ways, oes not act vate ang ogenes s an n ts tumor growt

Orthopedic Applications:

Overlaying stem cell-secreted ECM onto a decellularized, demineralized bone allograft could have a synergistic effect on bone healing, since osteogenic proteins are retained in the allograft (e.g. BMP -2) and give a more-than-additive cellular response in combination with ECM-resident proteins like VEGF (vascular endothelial growth factor) (Polo-Corrales et al. 2014). In addition, cultivation of cells on MSC-derived ECM has been shown in vitro to "prime" their responsiveness to exogenous growth signals (e.g. BMP -2) as shown through enhanced transcription of osteoinductive genes osteocalcin and bone sialoprotein (Lai et al. 2010).

In some embodiments, orthopedic applications of a homing ECM could be aided by the inclusion of growth factors and bioactive proteins known to be present in traditional cadaveric allograft tissue, such as demineralized bone matrix (DBM). These include bone morphogenetic proteins (BMPs), transforming growth factor beta (TGF-β), insulin-like growth factors I and II (IGF-I and IGF-II), PDGF, and basic and acidic fibroblast growth factor (bFGF and aFGF) (Solheim 1998). Other plant-derived compounds with bioactive effects in downstream cell- signaling pathways involved in bone development should also be considered. One such compound is honokiol, which shows considerable promise as a dual anabolic/anti-catabolic agent for the amelioration of multiple osteoporotic diseases (Yamaguchi et al. 2011). The role of the hematopoietic bone marrow niche in promoting development of bone cell precursors illustrates the importance, and possible inclusion of the factors angiopoietin-1, biglycan, osterix, and osteocalcin (Wu et al. 2009). The emerging importance of the collagen-binding bone developmental protein, osteopontin, in bone mineralization makes it a worthy candidate for infusion into the homECM (Shin et al. 2008).

Such compositions for enhanced bone healing may be used systemically or locally. Preferably, the compositions are administered locally.

General Wound-Healing Applications:

Where the disclosed compositions are used to enhance wound healing, the composition may comprise signaling markers that encourage re-epithelialization (for example TGF-P2), neovascularization (for example HIF-Ια), and/or fibroproliferation (for example PAI-1) (Arno et al. 2014). In some aspects, if the compositions are used for burn healing, the composition may further comprise an antimicrobial agent or the composition may be administered with an antimicrobial agent.

In some implementations, the composition may be infused with dermal matrix allografts for wound healing applications such as those in diabetic ulcer lesions or burn repair. In these implementations, the signaling marker of the composition comprises at least one of the group consisting of collagen IV, collagen V, fibronectin, laminin, GAG, EGFR, and PDGFR (Bielefield et al 2013). The GAG may be selected from the group consisting of hyaluronic acid and heparin sulfate.

In some implementations, the composition is applied to a dressing, for example on gauze, adhesive bandage, or mixed within liquid bandages. In some aspects, the disclosed compositions for wound healing may further comprise antimicrobial agents, which includes antibacterials, antifungals, antivirals, and antiparasitics. Examples of antimicrobial agents include antibiotics, benzoyl peroxide, azelaic acid, and oils of bay, cinnamon, clove, and thyme.

Such compositions for enhanced wound healing may be used systemically or locally. Cardiac Repair Applications:

Cardiac repair can be stimulated by inclusion in the homing ECM the signaling markers neuregulin-1 ( RG1) (Liang et al. 2015), VEGF, bFGF, HGF (Zhao et al. 2014), fibronectin and fibrinogen (Mayfield et al. 2014), etc. Cardiomyocyte function has been dramatically improved by coordinated release of Insulin-like Growth Factor (IGF) from the transplantation vehicle (Discher et al. 2009). OPN could be a very valuable candidate to include in the homing ECM considering that in vitro, OPN has been shown to stimulate vascular cell adhesion, migration, and survival. It is also thought to play a role in the homing and incorporation of EPCs (endothelial progenitor cells) to the site of endothelial injury, thus it may enhance EPC specific adhesion and function on biomaterial surfaces (Yuan et al. 2013). Additional pro-angiogenic factors previously considered for cardiac biomaterial engineering would include CEA-CAM1, versican, periostin, del-1, nephronectin, and the related protein EGFL-1 (Patra et al. 2015).

Such compositions for cardiac repair may be used systemically or locally. Cell Culture Applications:

The disclosed composition may also be used to regulate the growth and differentiation of cells in culture. In this application, the modified decellularized ECM could be laid on the bottom of tissue culture containers (for example multi-well trays or plates) prior to the addition of cells.

In one embodiment, the cells would be cultured for differentiation toward a particular lineage based on the modification of the decellularized ECM. Accordingly, the decellularized ECM may be modified with at least one signaling marker that would teach the cultured cells to differentiate in the direction of a transplantation target tissue. In some aspects, culturing cells over a layer of modified decellularized ECM could also expand the population of the cultured cells.

In other embodiments, rather than culturing patient tumor biopsy with fibroblast feeder cells, the growth of cells from the patient tumor biopsy could be enhanced by culturing theses cells over modified decellularized ECM. The decellularized ECM would be modified with additional compounds that induce "conditional reprogramming", such as the ROCK (Rho- kinase) inhibitor Y-27632, to provide the additional signals necessary to recapitulate the microenvironment experienced by the tumor prior to its removal. (Liu X, et al. 2012). In another embodiment, the decellularized ECM is made into a gel in order to provide a 3D jelly-like matrix for mimicking the tumor microenvironment of patient biopsies. The tumor is plated in the ECM gel, which, by virtue of the enriched growth factors, "wakes up" the tumor cells to resume their normal metabolic behavior. This allows for the expansion of the tumor cells in vitro. Expanded tumor cells can be replated in a multiwell assay plate to which anticancer compounds are applied, which enables screening of drug sensitivity. In some implementation, a special dye, such as one that fluoresces differently depending on what cellular pathways are disrupted, is applied along with the anticancer compound.

In another aspect, tumor cells expanded on the decellularized ECM gel are used for development of tumor specific antibodies. For example, the expanded tumor cells are injected into a rabbit or suitable host to generate an immune response and then the antibody sera is collected from their blood. The collected antibodies can be coupled to a fluorescent compound that would specifically illuminate the cancer cells. The collected antibodies can also be coupled to a lethal compound that would selectively target and kill the cancer cells. In some implementations, the collected antibody can be coupled to both a fluorescent compound and a lethal compound. Thus, the collected antibody provides for targeting for a specific type of tumor cells and enables the creation of a precision drug against the tumors cells.

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Claims

CLAIMS What Is Claimed Is:

1. A composition comprising a decellularized ECM (extracellular matrix) and at least one signaling marker, wherein the signaling marker is coupled to the decellularized ECM.

2. The composition of claim 1, wherein the decellularized ECM and the at least one signaling molecule are biotinylated.

3. The composition of claim 2, wherein the at least one signaling marker is coupled to the decellularized ECM by streptavidin.

4. The composition of claim 1, wherein the at least one signaling molecule is coupled to the decellularized ECM by amine-reactive succinimidyl esters.

5. The composition of any one of claims 1-4, wherein the at least one signaling marker is selected from the group consisting of a homing marker for the site of injury, a modifier of MSC gene expression, or both.

6. The composition of any one of claims 1-5, wherein the at least one signaling marker is a glycan.

7. The composition of claim 6, wherein the glycan is selected from the group consisting of VCAM and members of ICAM family.

8. The composition of any one of claims 1-5, wherein the at least one signaling marker is a MSC homing marker selected from the group consisting of SDF-Ια and related proteins.

9. The composition of any one of claims 1-5, wherein the at least one signaling marker is a tissue-specific growth factor.

10. The composition of any one of claims 1-5, wherein the at least one signaling marker is a growth factor selected from the group consisting of β-catenin family of proteins, BMP family of proteins, FGF family of proteins, PDGF family of proteins, and TGF superfamily of proteins.

11. The composition of claim 10, wherein the growth factor from the β-catenin family of proteins is Wnt.

12. The composition of claim 10, wherein the growth factor from the BMP family of proteins is selected from the group consisting of: BMP-1, BMP-2, BMP5, BMP7, and BMP8a.

13. The composition of claim 10, wherein the growth factor from the TGF superfamily of proteins is selected from the group consisting of: TGF-alpha, TGF-betal, and TGF-beta2.

14. The composition of any one of claims 1-13, wherein the decellularized ECM is isolated from a MSC culture.

15. The composition of any one of claims 1-13, wherein the decellularized ECM is isolated from a fibroblast culture.

16. The composition of claim 15, wherein the fibroblast culture comprises fibroblasts of a tissue origin selected from the group consisting of neural, epidermal, dermal, adipose, cardiac, kidney, muscle, liver, cartilage, pancreas, endometrium of uterus, umbilical cord, dental pulp, trabecular bone, and cortical bone.

17. The composition of any one of claims 1-16, wherein the decellularized ECM is isolated from a stromal cell culture.

18. The composition of claim 17, wherein the stromal cell culture comprises marrow stromal cells.

19. The composition of any one of claims 1-18, further comprising a dressing or a bandage, wherein the decellularized ECM is applied to the dressing or bandage.

20. The composition of claim 19, wherein the dressing or bandage is selected from the group consisting of: gauze, adhesive bandage, and liquid bandage.

21. The composition of any one of claims 1-18, further comprising demineralized bone matrix.

22. A method of targeting tissue engineering treatment to a site of injury comprising:

administering a modified decellularized ECM to a subject, wherein the modified decellularized ECM comprises a decellularized ECM coupled with a homing marker for the site of injury; and

administering to the subject MSCs.

23. The method of claim 22, wherein the decellularized ECM and the homing marker for the site of injury are biotinylated.

24. The method of claim 23, wherein a streptavidin molecule couples the homing marker for the site of injury to the decellularized ECM.

25. The method of claim 22, wherein amine-reactive succinimidyl esters couple the homing marker for the site of injury to the decellularized ECM.

26. The method of any one of claims 22-25, wherein the homing marker for the site of injury is a glycan.

27. The method of claim 26, wherein the glycan is selected from the group consisting of VCAM and members of ICAM family.

28. The method of any one of claims 22-27, wherein homing marker for the site of injury is a cytokine.

29. The method of claim 28, wherein the cytokine is selected from the group consisting of SDF- la and related proteins.

30. The method of any one of claims 22-29, wherein the ECM is administered systemically.

31. The method of any one of claims 22-29, wherein administering the modified decellularized ECM to a subject comprises applying a composition comprising the modified decellularized ECM and demineralized bone matrix to the site of injury.

32. The method of any one of claims 22-31, wherein the MSC is administered systemically.

33. The method of claim 30 or 31, wherein the MSC is administered locally.

34. A method of improving the therapeutic differentiation of MSCs in a subject comprising: administering to a subject a modified decellularized ECM, wherein the modified decellularized ECM comprises a decellularized ECM coupled with a modifier of MSC gene expression; and

administering to the subject MSCs.

35. The method of claim 34, wherein the decellularized ECM and the modifier of MSC gene expression are biotinylated.

36. The method of claim 34, wherein a streptavidin couples the modifier of MSC gene expression to the decellularized ECM.

37. The method of claim 34, wherein amine-reactive succinimidyl esters couple the homing marker for the site of injury to the decellularized ECM.

38. The method of any one of claims 34-37, wherein the modifier of MSC gene expression is a tissue-specific growth factor.

39. The method of any one of claims 34-37, wherein the modifier of MSC gene expression is a growth factor selected from the group consisting of β-catenin family of proteins, BMP family of proteins, FGF family of proteins, PDGF family of proteins, and TGF superfamily of proteins.

40. The method of claim 39, wherein the growth factor from the β-catenin family of proteins is Wnt.

41. The method of claim 39, wherein the growth factor from the BMP family of proteins is selected from the group consisting of: BMP-1, BMP-2, BMP5, BMP7, and BMP8a.

42. The method of claim 39, wherein the growth factor from the TGF superfamily of proteins is TGF-alpha, TGF-betal, and TGF-beta2.

43. The method of any one of claims 34-42, wherein administering the modified decellularized ECM to a subject comprises applying a composition comprising the modified decellularized ECM and demineralized bone matrix to the site of injury.

44. The method of any one of claims 34-43, wherein the MSC is administered systemically.

45. The method of any one of claims 34-43, wherein the MSC is administered locally.

46. The method of any one of claims 34-45, wherein the modified decellularized ECM further comprising a homing marker for the site of injury coupled to the decellularized ECM.

47. The method of claim 46, wherein the homing marker for the site of injury is a glycan selected from the group consisting of VCAM and members of ICAM family.

48. The method of claim 46, wherein the homing marker for the site of injury is a cytokine selected from the group consisting of SDF-Ια and related proteins.

49. A tumor cell-specific antibody composition comprising:

a fluorescent label; and

an anti-cancer antibody produced according a method comprising:

isolating tumor cells from a subject;

culturing the tumor cells with a composition accordingly to any one of claims 1-4, wherein the tumor cells isolated from the subject are expanded;

harvesting the expanded tumor cells;

administering the harvested expanded tumor cells to a host to induce an immune response in the host; and

harvesting serum from the host to extract antibodies against the expanded tumor cells, wherein the fluorescent label is coupled to the anti-cancer antibody.

50. The tumor cell-specific antibody composition of claim 49, further comprising a compound that selectively targets and kills the tumors cells isolated from the subject.

51. The tumor cell-specific antibody composition of claim 50, wherein the compound that selectively targets and kills the tumors cells isolated from the subject is selected by the method comprising:

isolating tumor cells from the subject;

culturing the tumor cells with a composition accordingly to any one of claims 1-4, wherein the tumor cells isolated from the subject are expanded;

administering an anti-cancer agent to the culture of expanded tumor cells; and

evaluating the growth of the culture of expanded tumor cells, wherein increased cell death in the culture of expanded tumor cells indicates that that anti-cancer agent kills the tumors cells isolated from the subject.

52. The tumor cell-specific antibody composition of claim 50 or 51, wherein the compound that selectively targets and kills the tumors cells isolated from the subject is coupled to the anticancer antibody.