WO2009080794A1

WO2009080794A1 - Method for preparing cell-specific extracellular matrices

Info

Publication number: WO2009080794A1
Application number: PCT/EP2008/068110
Authority: WO
Inventors: Marwan El-Sabban; Steen Sindet-Pedersen
Original assignee: MC2 Cell ApS
Current assignee: MC2 Cell ApS
Priority date: 2007-12-21
Filing date: 2008-12-19
Publication date: 2009-07-02
Anticipated expiration: 2010-06-21

Abstract

The invention is in the field of stem cell technology. This invention is directed to an in vitro method for inducing cells of human origin to produce cell-specific extracellular matrices and differentiating factors. This extract is suitable for use in differentiating human stem cells into desired tissue cells, such as differentiating bone marrow-derived stem cells into chondrocytes.

Description

METHOD FOR PREPARING CELL-SPECIFIC EXTRACELLULAR MATRICES FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Human tissues are endowed with a limited resident stem cell population that is engaged in the maintenance and limited repair of tissue injury. Under traumatic conditions, however, this population of resident cells is overwhelmed and efficient repair cannot be achieved, resulting in pathology. The signal for the stem cells to differentiate into organ-specific lineage is dependant, in the most part, on cell microenvironment comprising extracellular matrix (ecm)/cells and paracrine factors.

Current tissue engineering treatment modalities include the use of medical devices to support the function of the damaged tissue, pharmacologic supplementation of the metabolic products of the affected tissue, tissue transfer from a healthy site in the same or another individual. Problems with these current treatments include potential tissue complications and imperfect matches including the possible dependence on immunosuppressants, limited durability of the mechanical devices, and the inconvenience and complexity of pharmacologic supplementation. Current approaches for developing living tissue substitutes make use of a "scaffold" that serves as a physical support and template for cell attachment and tissue development. These scaffolds are ideally designed to resemble, both in structure and composition, the extracellular matrix that the cells are exposed to in vivo, in order to simulate the in vivo conditions. An early and widely used natural scaffold is made of the extracellular matrix protein collagen, while more recently, mechanically stronger artificial scaffolds made of substances such as poly-glycolic acid (PGA) and poly-lactic acid (PLA) have been used.

Some cell-scaffold compositions have multiple layers of biocompatible materials including extracellular matrix materials such as collagen, fibril-forming collagen, Matrix GIa protein, osteocalcin, or other biocompatible materials including marine coral, coralline hydroxyapatite, ceramic, and mixtures thereof, and some such scaffolds have been seeded with cells, and then placed within a bioreactor having a means for mechanically stimulating the cells at distinct frequencies (see U.S. Patent Application No. 20040005297 to P. R. Connelly et al, filed July 8, 2002, published January 8, 2004).

In addition, living tissue equivalents (LTEs), notably cell-seeded collagen and fibrin gels, have been used extensively as in vitro wound-healing models as well as systems for studying tissue remodeling. More recently, LTEs have begun to gain considerable attention as replacements for lost or damaged connective tissue (e.g., "Apligraf" from Organogenesis,

Inc.). LTEs have several advantages over synthetic alternatives including being a natural cell substrate, allowing cellularity to be achieved directly, and being conducive to cell spreading and extracellular matrix (ECM) formation. LTEs are made by mixing cells with a soluble biopolymer solution (e.g., collagen, fibrin, and/or proteoglycans). The cells invade, rearrange and partially degrade the biopolymer scaffold as well as synthesize new proteins throughout the culture period. However, LTEs generally lack the physical properties necessary to resist in vivo mechanical forces, and are not true "living tissues".

Over the last two decades, LTEs that are completely cell-derived have been developed. However, to date they have been very thin and taken a long time to grow, generally on the order of months, whereas collagen gels and fibrin gels can be developed in only a few days. There is a pressing need for complete biological cell-derived LTEs, and living scaffolds for use in wound repair and tissue regeneration in vitro and in vivo.

It is important here to highlight the weaknesses of existing preparations. For a start this is a generic matrix composition and not organ-specific. This, in the most part, is only scaffolding where the cells have to grow long enough to condition their environment to attain the proper differentiation pathway.

Recent studies have identified adult stem cells with the potential to differentiate into the specialized cells of unrelated tissues under certain conditions, including cells corresponding to a tissue derived from the same or from a different embryonic germ layer. See for example, Orlic et al. Nature 410: 701 -705 (2001 ), Gussoni et al. Nature 401 : 390-394 (1999), Bjornson et al., Science 283: 534-537 (1999). For example, blood stem cells (mesodermal origin) can under certain circumstances generate skeletal muscle cells (also mesodermal origin) and neurons (ectodermal origin).

Differentiation is a continuously regulated process and interactions between the cell and its environment play a major role in maintaining stable expression of differentiation-specific genes. An important component of the cellular environment is the extracellular matrix (ECM), which is composed of glycoproteins, proteoglycans and glycosaminoglycans that are secreted and assembled locally into an organized network to which cells adhere. An ECM is a component of the environment of all cell types, although the composition of the ECM and the spatial relationships between cells and ECM differ between tissues. Cells may be completely surrounded by ECM, as is the case for chondrocytes, or may contact the ECM only at one surface, as exemplified by epithelial and endothelial cells. In some tissues only a proportion of the cells are exposed to ECM: for example, in stratified epithelia.

While several tissue engineering breakthroughs have been made, there remain two important challenges to further progress in generating laboratory-grown tissues and organs: (1 ) the refinement of polymer scaffolding that mimics the organ architecture, and also supports the growth of appropriate stem cells; and (2) an abundant source of stem cells, i.e. those cells having the potential to proliferate and become fully specialized. Such cells, for example, can form bone, cartilage, muscle or fat, depending on the exact nature of their environment. Currently, most, if not all, organs and tissues made in the laboratory are generated using stem cells of animal or in some cases undefined human origin. Unfortunately, tissues made in this manner have very limited clinical use, primarily because they, like donor tissues and organs, are frequently rejected by the recipient's immune system. A scientifically sound and cost effective strategy to circumvent this problem is to use stem cells isolated from the intended tissue recipient.

Recent advances in cellular and molecular biology have created a window of opportunity for the successful isolation of stem cells from embryonic tissue, adult bone marrow, peripheral and umbilical cord blood.

Tissues and organs consist of specialized living cells arranged within a complex structural and functional framework of extracellular matrix (ECM). The great diversity observed in ECM composition contributes enormously to the properties and function of each organ and tissue: the rigidity and tensile strength of bone, the resilience of cartilage, the flexibility and hydrostatic strength of blood vessels, and the elasticity of skin, are examples of how different ECM compositions contribute to tissue function. Equally important is role of ECM during growth, development, and wound repair, where it provides a reservoir for soluble signaling molecules, and through its own dynamic composition, a source of additional signals to migrating, proliferating, and differentiating cells. These molecules are often referred to as soluble factors.

Artificial substitutes for ECM, called scaffolds, can consist of natural or synthetic polymers, or both, and have been used successfully alone and in combination with cells and soluble factors to induce tissue formation or promote tissue repair. Cells are also central to many tissue engineering strategies, and significant efforts have been made to identify and propagate pluhpotent stem cells, to identify signaling events important for proper differentiation, and to identify ideal micro-environments for maximum cellular function. These efforts that have led to a convergence of research in bioengineering, biomaterials,

ECM, cell growth and differentiation, and soluble factors that control cell fate.

The coordinated function of many cell types is regulated by the integration of extracellular signals derived from soluble factors such as growth factors, and insoluble molecules of the extracellular matrix (ECM). Indeed, accumulating data suggests that cellular behavior (for example growth, differentiation and cell migration) is regulated by the converging downstream signaling pathways of receptors for growth factors and ECM molecules. These findings have reinforced the importance of scaffold's composition and structure in controlling cellular responses in vitro and in vivo and provided a solid scientific foundation for the development of the new generation of biomaterials.

Based thereon such stem cells may therefore ultimately be used as a renewable source of cells that differentiate into a variety of tissue cells useful for treating a number of diseases and deficiencies. One important use is the treatment of neurological diseases such as Parkinson's disease ("PD"). Unfortunately, neural stem cells are not a particularly abundant source because they reside deep in the brain, severely constraining accessibility for harvesting. Conversely, bone marrow (BM) stem cells are more abundant and accessible.

The ease with which bone marrow stem cells are harvested by simple marrow aspiration, makes them excellent candidates for therapeutic use. BM comprises a number of stem cell types. Best known among these are hematopoietic stem cells (HSCs) and marrow stromal cells (MSCs). In normal mammals, HSCs give rise to blood cells whereas MSCs give rise to cell types that populate other tissues and sites such as cartilage or bone, hematopoietic supportive stromal cells and fat. Recent studies have suggested that these BM stem cells can, under appropriate conditions, differentiate into additional cell types such as cardiac myocytes, liver cells, and skeletal muscle cells.

Additionally, BM stem cells have been shown to have the potential for generating neurons (Sanchez-Ramos et al. Exp. Neurol. 164 247-256 (2000), Woodbury et al. J: Neurosci. Res. 62: 364-370 (2000), Mezey et al. Science 290: 1779-1782 (2000), Brazelton et al. Science 290: 775-1779 (2000). Chopp's group has investigated the use of human MSCs (hMSCs) to treat rats subjected to strokes. Li Y et al., Neurology, 2002, 59: 514-523, tested the effect of intravenously administered hMSCs on neurologic functional deficits after stroke. Treatment with hMSC resulted in significant recovery of function at 14 days compared with control rats with ischemia. Neurologic benefit resulting from this hMSC treatment appeared to derive from the increase of growth factors in the ischemic tissue, the reduction of apoptosis in the penumbral zone of the lesion, and the proliferation of endogenous cells in the subventricular zone. In a later publication from the same group, Chen X et al., J Neurosci Res, 2002, 69: 687-691 , investigated the temporal profile of various growth factors including brain- derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF), within cultures of human MSCs (hMSCs) conditioned with cerebral tissue extracts from traumatic brain injury (TBI). hMSCs in such cultures responded by producing more BDNF, NGF, VEGF, and HGF, supporting the notion that transplanted hMSCs provide therapeutic benefit in part via a responsive secretion of an array of growth factors that can foster neuro- protection and angiogenesis.

Laboratory grown cells derived from a several stem cell types, including BM-derived stem cells, may be a desirable source of transplantable material for grafting into brains of individuals suffering from neurological disorders.

To induce stem cells to differentiate, it is desirable to identify the right combination of molecules, their relative abundance and cell-culture conditions to (a) support survival and/or self-renewal of undifferentiated cells in culture and (b) stimulate them to become committed to a desired cell lineage. Such cells may then be implanted into an appropriate site in vivo to complete their growth and differentiation program. The process of HSC (or other stem cell) differentiation into particular progeny in vitro requires the action of many factors, including growth factors, extracellular matrix ("ECM") molecules and components, environmental stressors and direct cell-to-cell interactions. The appropriate agents that will enhance or direct stem cell differentiation along a particular path, however, may be difficult to predict.

For example, when human "leukemia inhibitory factor" (hLIF) was added to cultures of human MSCs, these cells developed fibroblastic morphologies (Sanchez-Romos et al.). The same protein, however, had been shown to be essential for maintaining mouse ES cells in an undifferentiated state (Sanchez et al., 2000). This illustrates the difficulty in knowing in advance the effect of a particular molecule on a particular cell type.

As appears from the above it is desirable to provide an extracellular matrix and soluble factors, perfectly reflecting the composition of the extracellular matrix of the tissue into which the stem cells are supposed to differentiate. Meanwhile the prior art does not teach a method by which a human-based extracellular matrix and paracrine factors may be produced without excising tissue from humans. In this context the above discussed LTEs are not suited for the production of extracellular matrix extracts, since the rate of secretion of extracellular matrix components is low in such differentiated cells.

SUMMARY OF THE INVENTION

The present invention is directed at the production of extracellular matrix components and soluble factors based on cultivating differentiating stem cells in order to induce their production of extracellular matrix. The present inventors have surprisingly found that human extracellular matrix extracts produced by differentiating stem cells largely reflect the corresponding in vivo composition of the extracellular matrix, and can thus later function as optimized differentiation environment for progenitors and stem cells, which will then differentiate into the cell types that are normally harboring the tissue having this extra cellular matrix composition.

According to the invention there is provided a method for producing an extracellular matrix extract and soluble factors of a desired human tissue comprising the steps:

• seeding human stem cells in a culture vessel in a medium comprising extracellular matrix resembling the extracellular matrix and soluble factors of the desired tissue, • culturing the cells in order to differentiate them into cells of the desired tissue, whereby the cells are stimulated to synthesize, secrete and organize extracellular matrix;

• continued culturing of the cells until the cells have been differentiated into cells of the desired tissue and have synthesized extracellular matrix and soluble factors; • collecting the soluble factors;

• removing the cells to obtain a cell free extracellular matrix extract of the desired tissue; and optionally mixing the soluble factors and cell free extracellular matrix extract.

Accordingly, the present invention provides a method for manufacturing extracellular extracts and soluble factors ex vivo, wherein the composition of the extracts resembles the human in vivo composition. Such extracts are very suitable for differentiating stem cells into the cells of a tissue of interest.

The extracellular matrix resembling the extracellular matrix of the desired tissue may be of human or animal origin, such as equine, canine, porcine, bovine, and ovine sources; or rodent species, such as mouse or rat. Although the aim of the present invention is to produce in vitro an extracellular matrix and soluble factors, which composition reflects the composition in a human being and thus essentially consists of growth factors, minerals etc secreted from human cells, an animal extracellular matrix extract may be used to induce the differentiation and thus stimulate the human stem cells to secrete an extracellular matrix of human origin. As the human stem cells differentiate they will produce an extracellular matrix layer or body, which predominantly contains differentiated human cells along with the extracellular matrix produced by them-selves; thus, if the stem cells have been stimulated to differentiate in an extract of animal origin there will be no fractions of non-human material left in the produced extracellular matrix layer. Since the in vivo composition of the extracellular matrix is achieved when the stem cells are fully differentiated into the cells of the tissue of interest it is necessary to cultivate the stem cells for generally at least 14 days.

In any event the cells are assessed before harvesting the extracellular matrix; as a general rule at least 90% of the cells should be fully differentiated before preparing the extracellular matrix.

If for some reason it is necessary to produce an extracellular matrix of a desired tissue not having any animal constituents the following alternatives are available: i) the stem cells are only initially stimulated with the animal derived extracellular matrix, thereafter the stimulated cells are carefully washed before they are cultured in a common growth medium; ii) the stem cells are stimulated with an extracellular matrix of human origin (difficult to obtain due to pain and regulatory hurdles), and iii) the stem cells are stimulated with a "synthetic" extracellular matrix comprising growth and differentiation factors known to be (at least partially) responsible for the differentiation of stem cells into a given tissue cell type.

The present invention is further directed to the use of extracts according to the present invention for differentiation of stem cells. For example the extracts may be used for differentiation of mesenchymal stem cells into at least one type of tissue, in particular into bone or cartilage tissue. Likewise the extracts may be used for differentiation of hematopoietic stem cells into hematopoietic progenitor cells.

In one aspect, the present method is used to manufacture extracellular matrix pertaining to cartilage tissue by seeding and culturing stem cells in a medium resembling the composition of the extracellular matrix of cartilage tissue for stimulated synthesis of cartilage extracellular matrix extract. This ECM extract can be directly applied to adult stem cells to differentiate them into chondrocytes.

Accordingly the present invention also provides a method for differentiating human bone marrow-derived stem cells into chondrocytes, comprising:

• providing bone marrow-derived stem cells from a human;

• treating the stem cells with a cartilage extracellular matrix extract of the present invention; and

• cultivating and differentiating the stem cells until a sufficient amount of chondrocytes have been obtained.

In another aspect, the present method is used to manufacture extracellular matrix pertaining to liver tissue by seeding and culturing hepatocytes for stimulated synthesis of extracellular matrix extract resembling the composition of the matrix in liver tissue. This ECM extract can be directly applied to adult stem cells to differentiate them into hepatocytes. Accordingly the present invention also provides a method for differentiating bone human marrow-derived stem cells into hepatocytes, comprising:

• providing bone marrow-derived stem cells from a human;

• treating the stem cells with a liver extracellular matrix extract of the present invention; and • cultivating and differentiating the stem cells until a sufficient amount of hepatocytes have been obtained. In still another aspect, the present method is used to manufacture extracellular matrix pertaining to pancreas tissue, particularly islets tissue, by seeding and culturing pancreas beta-cells for stimulated synthesis of extracellular matrix extract resembling the composition of the matrix in islet tissue. This ECM extract can be directly applied to adult stem cells to differentiate them into beta cells. Accordingly the present invention also provides a method for differentiating bone human marrow-derived stem cells into beta-cells, comprising:

• providing bone marrow-derived stem cells from a human;

• treating the stem cells with a pancreas extracellular matrix extract of the present invention; and • cultivating and differentiating the stem cells until a sufficient amount of beta- cells have been obtained.

Despite the potential benefit of using human embryonic stem cells in the treatment of disease, their use remains controversial because of their derivation from early embryos. In contrast, adult stem cells are found in all tissues of the body and have the potential to differentiate into multitude of cell types. The specific advantage of using adult stem cells for tissue repair is that the patient's own cells could be expanded in vitro and then reintroduced into the patient. This autologous grafting elevates the use of immune-suppressants and hence reduces the risk possibility of infection and immune rejection. Thus, the goal of stem cell therapy is to repair the damaged tissues that are inefficient in their ability to heal themselves. Currently, replacing diseased or destroyed organs relied mainly on donation and transplantation. Stem cell therapy offers an effective and less invasive alternative to treat bone fractures, or enhance the repair of tissues that require prolonged time to fully heal.

Bone marrow-derived mesenchymal stem cells (BMMSC) are a population of adult multipotent stem cells located within the bone marrow. These cells are characterized by their ability to differentiate into at least three phenotypes (chondrocytes, osteocytes and adipocytes) when cultured in vitro (Jaiswal 2000 and Pittenger 1999). Once uncommitted mesenchymal stem cells are isolated and purified from the bone marrow they maintain the capacity to self-renew and differentiate along multiple pathways resulting in the generation and maintenance of a variety of tissues. This has advanced the possibility of utilizing these cells to repair or replace damaged tissues. Previous studies suggest that differentiation of mesenchymal stem cells is modulated by various growth factors and cytokines, most of which belong to the transforming growth factor β superfamily. Moreover, many of these growth factors, such as TGF-β1 , were clearly shown to be involved in chondrogenic differentiation. On the other hand, the extracellular matrix (ECM) plays an essential role in BMMSC differentiation. The ECM provides cells with mechanical support and cyto-architecture as well as components involved cellular signaling to induce and maintain a differentiating phenotype. Differentiating cells monitor the composition and production of the ECM it is secreting to accommodate the changing mechanical needs of differentiating cell type, for example chondrocytes.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows negative cytotoxic effect of cartilage matrix extracts on cultured BMMSCs (A). Decrease in cellular proliferation as an indication of onset of differentiation: BMMSC were treated with cartilage matrix extracts. Cellular proliferation was monitored at day 1 , day 3, day 7 and day 14 post treatment. Upon treatment with organ-derived extracts there was a significant decrease in cellular proliferation. Untreated BMMSC maintained a normal proliferation rate (B).

Figure 2 shows the expression of Sox9 using real time PCR technique: Joint and meniscus extracts induce chondrogenic differentiation of BMMSCs through the upregulation of Sox9 mRNA (A).

Figure 3 shows the expression of Collagen type I using real time PCR technique. Collagen type I, which is known to be upregulated during osteogenic differentiation, was not detected (B).

Figure 4 shows aggrecan expression: Real-time PCR revealed decreased levels of aggrecan mRNA (suggesting late onset of aggrecan expression) (A).

Figure 5 relates to aggrecan positive cells, which showed major increase in aggrecan positive cells (C). Figure 6 shows morphological changes during BMMSC chondrocytic differentiation 14 days post treatment: showing aggregates (arrows) with central alignment of hypertrophic cells. 10ng/ml TGF-β1 recombinant protein was used as positive control.

Figure 7 shows bovine and equine bone extracts (Colloss^® and Colloss-E^®) induce in vitro differentiation of BMMSCs. BMMSCs at passage six were treated with a 1 :50 dilution of Colloss (bovine) or Colloss-E (equine) (28 and 32 μg/ml, respectively) for different time points. (A) Morphological changes of BMMSCs induced by treatment with Colloss and Colloss-E for 7 days (n = 6). (B) Hematoxylin and eosin staining of the BMMSC cell culture after treatment with Colloss and Colloss-E for 15 days (n = 3). (C) Proliferation of BMMSC at different time intervals after treatment with Colloss and Colloss-E (n= 6).

Figure 8 shows that Colloss^® and Colloss-E^® induce alkaline phosphatase activity in BMMSCs. BMMSCs at passage six were treated with a 1 :50 dilution of Colloss (bovine) or Colloss-E (equine) (28 and 32 μg/ml, respectively) for 7 or 14 days. (A) Analysis of alkaline phosphatase protein expression by western blot on medium-free cell extracts in the absence or presence of Colloss or Colloss-E (n = 2). (B) Alkaline phosphatase enzyme activity was measured in medium-free cell extracts in the absence or presence of Colloss or Colloss-E at day 7 and day 14. Results are expressed as U/ml/h/μg protein (n = 2).

Figure 9 shows that Colloss^® and Colloss-E^® induce osteopontin expression in BMMSCs. BMMSCs at passage six were treated with a 1 :50 dilution of Colloss (bovine) or Colloss-E (equine) (28 and 32 μg/ml, respectively) for 7, 14 and 30 days. (A) Osteopontin expression was assessed by immunofluorescence using FITC-conjugated secondary antibodies. Untreated control cells show a positive baseline osteopontin expression (left), while treated cells exhibited increased signal intensity (right). (B) The amount of secreted osteopontin in day 30 cell-free supernatant from cultured BMMSCs was assessed by ELISA, as described in material and methods (n = 4). Statistics represent comparison of Colloss or Colloss-E with untreated control. ^*p < 0.05; ^**p < 0.01 . BMMSC: Bone marrow-derived mesenchymal stem cell.

Figure 10 shows that Colloss^® and Colloss-E^® induce calcium deposition in BMMSCs.

BMMSC at passage six were treated with a 1 :50 dilution of Colloss (bovine) or Colloss-E

(equine) (28 and 32 μg/ml, respectively) for 15 days. (A) Calcium deposits were visualized at day 15 in H&E-stained BMMSCs. (B) Quantification of calcium deposits was achieved by counting total calcium deposits on a coverslip and expressed as deposits/100 mm², (n = 3). Statistics represent comparison of Colloss or Colloss-E to untreated control: ^*p < 0.05. BMMSC: Bone marrow-derived mesenchymal stem cell.

Figure 1 1 shows typical hepatocyte markers induced in BMMSC

Figure 12 shows the induction of p450 expression in ECM differentiated BMMSCs.

Figure 13 shows the morphology of differentiated BMMSCs cultivated on human liver cell ECM.

Figure 14 shows expression of Liver-specific connexins in differentiated stem cells (hepatocytes).

Figure 15 shows Induction of p450 in liver-differentiated BMMSC.

Figure 16 shows glucose uptake in liver-differentiated BMMSC compared to undifferentiated BMMSC.

DETAILED DESCRIPTION OF THE INVENTION

Current engineered living tissue constructs are not completely cell assembled and must rely on either the addition or incorporation of exogenous matrix components or synthetic members for structure or support, or both.

The culture media or extracts of the present invention exhibit many of the native features of the tissue from which their cells are derived.

Definitions

"Resembles" as used herein means there is physical, compositional, structural, functional, phenotypic or other similarities between the materials or systems being compared, such that the objects are substantially equivalent. "Substantially equivalent" means that visible, microscopic, physical, functional, and other observations and assays do not easily or significantly distinguish the materials or systems. An easy or significant distinction would, for example, be a functional difference, a physical difference, a compositional difference, a structural difference immediately apparent, or easily detectable with standard assays and observational techniques such as staining, microscopy, antibodies, etc. "Extracellular Matrix" (ECM) or "Cell Derived Matrix" (CDM) or Cell-produced Matrix as used interchangeably herein means a cell-derived secreted substance produced by and/or secreted from cells into the extracellular space. The ECM/CDM provides a growth template for any cell type to grow, differentiate, and produce tissue.

The ECM allows cell attachment and cell migration, and promotes cell differentiation. The ECM also aids the formation of new tissue of a desired or existing cell type. As used herein

"Cell-Produced Matrix, also called Cell-Derived Matrix (CDM)" also means a 3-dimensional ECM (or matrix) structure that has been completely produced and arranged by cells (or entities) in vitro.

"Construct" as used herein means a physical structure with mechanical properties such as a matrix of scaffold. Construct encompasses both autogenic living scaffolds and living tissue matrices, ex-vivo cell- produced tissue and cell-derived matrix. "Cell-derived" as used herein means that the source for the material, body, or component is a cell or a collection of cells.

"Ex-vivo Cell-produced Tissue (ECT)" as used herein means, a functional tissue comprising one or more types of cells (or entities) and the ECM (or matrix) that has been completely produced and arranged by some of these cells (or entities). "Living Tissue Matrix (LTM)" as used herein means, a 3-dimensional tissue (or matrix) that is capable of being transformed into a more complex tissue (or matrix) or a completely different type of tissue (or matrix) that consists of cells (or entities) and the ECM (or matrix) that has been completely produced and arranged by these cells (or entities).

"Living Tissue Equivalent (LTE)" as used herein means a construct containing living cells that intends to mimic a certain type of native tissue. This construct can be produced by any means in vitro, including by the use of artificial scaffolds.

"Culturing the cells in order to differentiate them" as used herein, means conditions that facilitate, aid, further or in any way allow the development of three-dimensional tissue growth. Conditions may include use of specific media, growth factors, minerals, incubation temperature, cell density, aeration, agitation, use of ALS "molds" to shape and contain growth of desired tissue, use of sub-atmospheric pressure chambers such as Synthecon's near-zero-gravity incubator systems (such as HARVs and STLVs) for growth of desired tissue, use of micro-carrier beads, use of natural or biodegradable scaffolds, implanting a non-fibroblast-seeded autogenic living scaffold within an in vivo site such as in an organ or tissue such as connective, epithelial, muscle, and/or nerve tissue.

"Genetically engineered" as used herein means that a cell or entity, by human manipulation such as chemical, physical, stress-induced, or other means, has undergone mutation and selection; or that an exogenous nucleic acid has actually been introduced to the cell or entity through any standard means, such as transfection; such that the cell or entity has acquired a new characteristic, phenotype, genotype, and/or gene expression product, including but not limited to a gene marker, a gene product, and/or a mRNA, to endow the original cell or entity, at a genetic level, with a function, characteristic, or genetic element not present in non-genetically engineered, non-selected counterpart cells or entities.

Preferred embodiments

Cells originating from liver (hepatocytes) produce extracellular matrix components different from cells originating from e.g. skin and connective tissue (fibroblast). Fibroblast cells, generally, produce a number of extracellular matrix proteins, primarily collagen. There are several types of collagens produced by fibroblasts, however, type I collagen is the most prevalent in vivo. Human fibroblast cell strains can be derived from a number of sources, including, but not limited to neonate male foreskin, dermis, tendon, lung, umbilical cords, cartilage, urethra, corneal stroma, oral mucosa, and intestine. The human cells may include but need not be limited to fibroblasts, but may include: smooth muscle cells, chondrocytes and other connective tissue cells of mesenchymal origin. It is preferred, but not required, that the origin of the matrix-producing cell used in the production of a tissue construct be derived from a tissue type that it is to resemble or mimic after employing the culturing methods of the invention. For instance, where a skin-construct is produced, the matrix- producing cell is a fibroblast, preferably of dermal origin. In another preferred embodiment, fibroblasts isolated by microdissection from the dermal papilla of hair follicles can be used to produce the matrix alone or in association with other fibroblasts. In the embodiment where a corneal-construct is produced, the matrix-producing cell is derived from corneal stroma. Cell donors may vary in development and age. Cells may be derived from donor tissues of embryos, neonates, or older individuals including adults. Surprisingly adult stem cells (e.g. bone marrow derived stem cells) are differentiated to develop into the desired cell type by culturing them in the extracts of the present invention.

Although human cells are preferred for use in the invention, the cells to be used in the method of are not limited to cells from human sources. Cells from other mammalian species including, but not limited to, equine, canine, porcine, bovine, and ovine sources; or rodent species such as mouse or rat may be used. In addition, cells that are spontaneously, chemically or virally transfected or recombinant cells or genetically engineered cells may also be used in this invention. For those embodiments that incorporate more than one cell type, chimeric mixtures of normal cells from two or more sources; mixtures of normal and genetically modified or transfected cells; or mixtures of cells of two or more species or tissue sources may be used.

The matrix-producing cell is cultured in a vessel suitable for animal cell or tissue culture, such as a culture dish, flask, or roller-bottle, which allows for the formation of a three- dimensional tissue-like structure. Suitable cell growth surfaces on which the cells can be grown can be any biologically compatible material to which the cells can adhere and provide an anchoring means for the cell-matrix construct to form. Materials such as glass; stainless steel; polymers, including polycarbonate, polystyrene, polyvinyl chloride, polyvinylidene, polydimethylsiloxane, fluoropolymers, and fluorinated ethylene propylene; and silicon substrates, including fused silica, polysilicone, or silicon crystals may be used as a cell growth surfaces.

While the tissue construct of the invention may be grown on a solid cell growth surface, a cell growth surface with pores that communicate both top and bottom surfaces of the membrane to allow bilateral contact of the medium to the developing tissue construct or for contact from only below the culture is preferred. Bilateral contact allows medium to contact both the top and bottom surfaces of the developing construct for maximal surface area exposure to the nutrients contained in the medium. Medium may also contact only the bottom of the forming cultured tissue construct so that the top surface may be exposed to air, as in the development of a cultured skin construct. The preferred culture vessel is one that utilizes a carrier insert, a culture-treated permeable member such as a porous membrane that is suspended in the culture vessel containing medium. Typically, the membrane is secured to one end of a tubular member or framework that is inserted within and interfaces with a base, such as a petri or culture dish that can be covered with a lid. When these types of culture vessels are employed, the tissue-construct is produced on one surface of the membrane, preferably the top, upwardly facing surface and the culture is contacted by cell media on both top and bottom surfaces. The pores in the growth surface allow for the passage of culture media for providing nutrients to the underside of the culture through the membrane, thus allowing the cells to be fed bilaterally or solely from the bottom side. A preferred pore size is one that is small enough that it does not allow for the growth of cells through the membrane, yet large enough to allow for free passage of nutrients contained in culture medium to the bottom surface of the cell-matrix construct, such as by capillary action. Preferred pore sizes are about less than 3 microns but range between about 0.1 microns to about 3 microns, more preferably between about 0.2 microns to about 1 micron and most preferably about 0.4 micron to about 0.6 micron sized pores are employed. In the case of human dermal fibroblasts, the most preferred material is polycarbonate having a pore size is between about 0.4 to about 0.6 microns. The maximum pore size depends not only on the size of the cell but also the ability of the cell to alter its shape and pass through the membrane. It is important that the tissue-like construct adheres to the surface but does not incorporate or envelop the substrate so it is removable from it such as by peeling with minimal force. The size and shape of the tissue construct formed is dictated by the size of the vessel surface or membrane on which it grown. Substrates may be round or angular or shaped with rounded corner angles, or irregularly shaped. Substrates may also be flat or contoured as a mold to produce a shaped construct to interface with a wound or mimic the physical structure of native tissue. To account for greater surface areas of the growth substrate, proportionally more cells are seeded to the surface and a greater volume of media is needed to sufficiently bathe and nourish the cells. When the tissue construct is finally formed, whether it is a single layer cell-matrix construct or a bi-layer construct, it is removed by peeling from the membrane substrate before grafting to a patient.

The system for the production of the cell-matrix layer may be either static or may employ a perfusion means to the culture media. In the static system, the culture medium is still and relatively motionless as contrasted to the perfusion system where the medium is in motion. The perfusion of medium affects the viability of the cells and augments the development of the matrix layer. Perfusion means include, but are not limited to: using a magnetic stir bar or motorized impeller in the culture dish subjacent (below) or adjacent to the substrate carrier containing the culture membrane to stir the medium; pumping medium within or through the culture dish or chamber; gently agitating the culture dish on a shaking or rotating platform; or rolling, if produced in a roller bottle. Other perfusion means can be determined by one skilled in the art for use in the method of the invention. Culture media formulations suitable for use in the present invention are selected based on the cell types to be cultured and the extracellular matrix to be produced. The culture medium that is used and the specific culturing conditions needed to promote cell growth, matrix synthesis, and viability will depend on the type of cell being grown.

The use of chemically defined culture media is preferred, that is, media free of undefined animal organ or tissue extracts, for example, serum, pituitary extract, hypothalamic extract, placental extract, or embryonic extract or proteins and factors secreted by feeder cells. In a most preferred embodiment, the media are free of undefined components and defined biological components derived from non-human sources. When the invention is carried out utilizing screened human cells cultured using chemically defined components derived from no non-human animal sources, the resultant tissue construct is a defined human tissue construct. Synthetic functional equivalents may also be added to supplement chemically defined media within the purview of the definition of chemically defined for use in the most preferred fabrication method. Generally, one of skill in the art of cell culture will be able to determine suitable natural human, human recombinant, or synthetic equivalents to commonly known animal components to supplement the culture media of the invention without undue investigation or experimentation.

The advantages in using such a construct in the clinic is that the concern of adventitious animal or cross-species virus contamination and infection is diminished. In the testing scenario, the advantages of a chemically defined construct is that when tested, there is no chance of the results being confounded due to the presence of the undefined components.

Culture medium is comprised of a nutrient base usually further supplemented with other components. The skilled scientist can determine appropriate nutrient bases in the art of animal cell culture with reasonable expectations for successfully producing a tissue construct of the invention. Many commercially available nutrient sources are useful on the practice of the present invention. These include commercially available nutrient sources which supply inorganic salts, an energy source, amino acids, and B-vitamins such as Dulbecco's Modified Eagle's Medium (DMEM); Minimal Essential Medium (MEM); M199; RPMI 1640; Iscove's Modified Dulbecco's Medium (EDMEM). Minimal Essential Medium (MEM) and M199 require additional supplementation with phospholipid precursors and nonessential amino acids. Commercially available vitamin-rich mixtures that supply additional amino acids, nucleic acids, enzyme cofactors, phospholipid precursors, and inorganic salts include Ham's F-12, Ham's F-10, NCTC 109, and NCTC 135. Albeit in varying concentrations, all basal media provide a basic nutrient source for cells in the form of glucose, amino acids, vitamins, and inorganic ions, together with other basic media components. The most preferred base medium of the invention comprises a nutrient base of either calcium-free or low calcium Dulbecco's Modified Eagle's Medium (DMEM), or, alternatively , DMEM and Ham's F-12 between a 3-to-1 ratio to a 1 -to-3 ratio, respectively.

The base medium is supplemented with components such as amino acids, growth factors, and hormones. Defined culture media for the culture of cells of the invention are described in United States Patent No. 5,712,163 and in International PCT Publication No. WO 95/31473 the disclosures of which are incorporated herein by reference. Other media are known in the art such as those disclosed in Ham and McKeehan, Methods in Enzymology,

58:44-93 (1979), or for other appropriate chemically defined media, in Bottenstein et al., Methods in Enzymology, 58:94-109 (1979). In the preferred embodiment, the base medium is supplemented with the following components known to the skilled artisan in animal cell culture: insulin, transferrin, triiodothyronine (T3), and either or both ethanolamine and o- phosphoryl-ethanolamine, wherein concentrations and substitutions for the supplements may be determined by the skilled artisan.

Insulin is a polypeptide hormone that promotes the uptake of glucose and amino acids to provide long term benefits over multiple passages. Supplementation of insulin or insulin-like growth factor (IGF) is necessary for long term culture as there will be eventual depletion of the cells' ability to uptake glucose and amino acids and possible degradation of the cell phenotype. Insulin may be derived from either animal, for example bovine, human sources, or by recombinant means as human recombinant insulin. Therefore, a human insulin would qualify as a chemically defined component not derived from a non-human biological source. Insulin supplementation is advisable for serial cultivation and is provided to the media at a wide range of concentrations. A preferred concentration range is between about 0.1 μg/ml to about 500 μg/ml, more preferably at about 5 μg/ml to about 400 μg/ml, and most preferably at about 375 μg/ml. Appropriate concentrations for the supplementation of insulin-like growth factor, such as IGF-1 or IGF-2, may be easily determined by one of skill in the art for the cell types chosen for culture.

Transferrin is in the medium for iron transport regulation. Iron is an essential trace element found in serum. As iron can be toxic to cells in its free form, in serum it is supplied to cells bound to transferrin at a concentration range of preferably between about 0.05 to about 50 μg/ml, more preferably at about 5 μg/ml. Triiodothyronine (T3) is a basic component and is the active form of thyroid hormone that is included in the medium to maintain rates of cell metabolism. Truodothyronine is supplemented to the medium at a concentration range between about 0 to about 400 pM, more preferably between about 2 to about 200 pM and most preferably at about 20 pM.

Either or both ethanolamine and o-phosphoryl-ethanolamine, which are phospholipids, are added whose function is an important precursor in the inositol pathway and fatty acid metabolism. Supplementation of lipids that are normally found in serum is necessary in a serum-free medium. Ethanolamine and o-phosphoryl-ethanolamine are provided to media at a concentration range between about 10^'6 to about 10^'2 M, more preferably at about 1 x 10^'4 M.

Throughout the culture duration, the base medium is additionally supplemented with other components to induce synthesis or differentiation or to improve cell growth such as hydrocortisone, selenium, and L-glutamine.

Hydrocortisone has been shown in keratinocyte culture to promote keratinocyte phenotype and therefore enhance differentiated characteristics such as involucrin and keratinocyte transglutaminase content (Rubin et al., J. Cell PhysioL, 138:208-214 (1986)). Therefore, hydrocortisone is a desirable additive in instances where these characteristics are beneficial such as in the formation of keratinocyte sheet grafts or skin constructs. Hydrocortisone may be provided at a concentration range of about 0.01 ug/ml to about 4.0 μg/ml, most preferably between about 0.4 μg/ml to 16 ug/ml.

Selenium is added to serum-free media to resupplement the trace elements of selenium normally provided by serum. Selenium may be provided at a concentration range of about 10^~9 M to about 10^~7 M; most preferably at about 5.3 x 10^"8 M.

The amino acid L-glutamine is present in some nutrient bases and may be added in cases where there is none or insufficient amounts present. L-glutamine may also be provided in stable form such as that sold under the mark, GlutaMAX-1 ™ (Gibco BRL, Grand Island,

NY). GlutaMAX-1 ™ is the stable dipeptide form of L-alanyl-L-glutamine and may be used interchangeably with L-glutamine and is provided in equimolar concentrations as a substitute to L-glutamine. The dipeptide provides stability to L-glutamine from degradation over time in storage and during incubation that can lead to uncertainty in the effective concentration of L-glutamine in medium. Typically, the base medium is supplemented with preferably between about 1 mM to about 6 mM, more preferably between about 2 mM to about 5 mM, and most preferably 4 mM L-glutamine or GlutaMAX-1 ™.

Growth factors such as epidermal growth factor (EGF) may also be added to the medium to aid in the establishment of the cultures through cell scale-up and seeding. EGF in native form or recombinant form may be used. Human forms, native or recombinant, of EGF are preferred for use in the medium when fabricating a skin equivalent containing no non- human biological components. EGF is an optional component and may be provided at a concentration between about 1 to 15 ng/mL, more preferably between about 5 to 10 ng/mL.

The medium described above is typically prepared as set forth below. However, it should be understood that the components of the present invention may be prepared and assembled using conventional methodology compatible with their physical properties. It is well known in the art to substitute certain components with an appropriate analogous or functionally equivalent acting agent for the purposes of availability or economy and arrive at a similar result. Naturally occurring growth factors may be substituted with recombinant or synthetic growth factors that have similar qualities and results when used in the performance of the invention.

Media in accordance with the present invention are sterile. Sterile components are bought sterile or rendered sterile by conventional procedures, such as filtration, after preparation. Proper aseptic procedures were used throughout the following Examples. DMEM and F-12 are first combined and the individual components are then added to complete the medium.

Stock solutions of all components can be stored at -20 ⁰C., with the exception of nutrient source that can be stored at 4 ⁰C. All stock solutions are prepared at 500X final concentrations listed above. A stock solution of insulin, transferrin and triiodothyronine (all from Sigma) is prepared as follows: triiodothyronine is initially dissolved in absolute ethanol in IN hydrochloric acid (HCI) at a 2:1 ratio. Insulin is dissolved in dilute HCI (approximately

0.1 N) and transferrin is dissolved in water. The three are then mixed and diluted in water to a 500X concentration. Ethanolamine and o-phosphoryl-ethanolamine are dissolved in water to 500X concentration and are filter sterilized. Progesterone is dissolved in absolute ethanol and diluted with water. Hydrocortisone is dissolved in absolute ethanol and diluted in phosphate buffered saline (PBS). Selenium is dissolved in water to 500X concentration and filter sterilized. EGF is purchased sterile and is dissolved in PBS. Adenine is difficult to dissolve but may be dissolved by any number of methods known to those skilled in the art. Serum albumin may be added to certain components in order to stabilize them in solution and are presently derived from either human or animal sources. For example, human serum albumin (HSA) or bovine serum albumin (BSA) may be added for prolonged storage to maintain the activity of the progesterone and EGF stock solutions. The medium can be either used immediately after preparation or, stored at 4 ⁰C. If stored, EGF should not be added until the time of use.

In order to form the cell-matrix layer by the culture of matrix-producing cells, the medium is supplemented with additional agents that promote matrix synthesis and deposition by the cells. These supplemental agents are cell-compatible, defined to a high degree of purity and are free of contaminants. The medium used to produce the cell-matrix is termed "matrix production medium".

To prepare the matrix production medium, the base medium is supplemented with an ascorbate derivative such as sodium ascorbate, ascorbic acid, or one of its more chemically stable derivatives such as L-ascorbic acid phosphate magnesium salt n-hydrate. Ascorbate is added to promote hydroxylation of proline and secretion of procollagen, a soluble precursor to deposited collagen molecules. Ascorbate has also been shown to be an important cofactor for post-translational processing of other enzymes as well as an upregulator of type I and type III collagen synthesis.

While not wishing to be bound by theory, supplementing the medium with amino acids involved in protein synthesis conserves cellular energy by not requiring the cells produce the amino acids themselves. The addition of proline and glycine is preferred as they, as well as the hydroxylated form of proline, hydroxyproline, are basic amino acids that make up the structure of collagen.

While not required, the matrix-production medium is optionally supplemented with a neutral polymer. The cell-matrix constructs of the invention may be produced without a neutral polymer, but again not wishing to be bound by theory, its presence in the matrix production medium may collagen processing and deposition more consistently between samples. One preferred neutral polymer is polyethylene glycol (PEG), which has been shown to promote in vitro processing of the soluble precursor procollagen produced by the cultured cells to matrix deposited collagen. Tissue culture grade PEG within the range between about 1000 to about 4000 MW (molecular weight), more preferably between about 3400 to about 3700

MW is preferred in the media of the invention. Preferred PEG concentrations are for use in the method may be at concentrations at about 5% w/v or less, preferably about 0.01 % w/v to about 0.5% w/v, more preferably between about 0.025% w/v to about 0.2% w/v, most preferably about 0.05% w/v. Other culture grade neutral polymers such dextran, preferably dextran T-40, or polyvinylpyrrolidone (PVP), preferably in the range of 30,000-40,000 MW, may also be used at concentrations at about 5% w/v or less, preferably between about 0.01% w/v to about 0.5% w/v, more preferably between about 0.025% w/v to about 0.2% w/v, most preferably about 0.05% w/v. Other cell culture grade and cell-compatible agents that enhance collagen processing and deposition may be ascertained by the skilled routineer in the art of mammalian cell culture.

When the cell producing cells are confluent, and the culture medium is supplemented with components that assist in matrix synthesis, secretion, or organization, the cells are said to be stimulated to form a tissue-construct comprised of cells and matrix synthesized by those cells. Therefore, a preferred matrix production medium formulation comprises: a base 3:1 mixture of Dulbecco's Modified Eagle's Medium (DMEM) (high glucose formulation, without L-glutamine) and Hams F-12 medium supplemented with either 4 mM L-glutamine or equivalent, 5 ng/ml epidermal growth factor, 0.4 μg/ml hydrocortisone, 1 x 10^'4 M ethanolamine, 1 x 10^'4 M o-phosphoryl-ethanolamine, 5 μg/ml insulin, 5 μg/ml transferrin, 20 pM triiodothyronine, 6.78 ng/ml selenium, 50 ng/ml L-ascorbic acid, 0.2 μg/ml L-proline, and 0.1 μg/ml glycine. To the production medium, other pharmacological agents may be added to the culture to alter the nature, amount, or type of the extracellular matrix secreted. These agents may include polypeptide growth factors, transcription factors or inorganic salts to up- regulate collagen transcription. Examples of polypeptide growth factors include transforming growth factor-beta 1 (TGF-β1 ) and tissue-plasmmogen activator (TPA), both of which are known to upregulate collagen synthesis. Raghow et al., Journal of Clinical Investigation, 79:1285-1288 (1987); Pardes et al., Journal of Investigative Dermatology, 100:549 (1993). An example of an inorganic salt that stimulates collagen production is cerium. Shivakumar et al., Journal of Molecular and Cellular Cardiology 24:775-780 (1992).

The cultures are maintained in an incubator to ensure sufficient environmental conditions of controlled temperature, humidity, and gas mixture for the culture of cells. Preferred conditions are between about 34 ⁰C to about 38 ⁰C, more preferably 37 ± 1 ⁰C with an atmosphere between about 5-10 ± 1 % CO₂ and a relative humidity (Rh) between about 80-

90%.

Once sufficient cell numbers have been obtained, cells are harvested and seeded onto a suitable culture surface and cultured under appropriate growth conditions to form a confluent sheet of cells. In the preferred embodiment, the cells are seeded on a porous membrane that is submerged to allow medium contact from below the culture through the pores and directly above. Preferably, cells are suspended in either base or growth media and are seeded on the cell culture surface at a density between about 1 x 10⁵ cells/cm² to about 6.6 x 10⁵ cells/cm², more preferably between about 3 x 10⁵ cells/cm² to about 6.6 x

10⁵ cells/cm² and most preferably at about 6.6 x 10⁵ cells/cm² (cells per square centimeter area of the surface). Cultures are cultured in growth medium to establish the culture and are cultured to between about 80% to 100% confluence at which time they are induced chemically by changing the medium to matrix production medium in order to upregulate the synthesis and secretion of extracellular matrix. In an alternate method, cells are seeded directly in production media to eliminate the need to change from the basic media to the production media but it is a method that requires higher seeding densities.

During the culture, the cells organize the secreted matrix molecules to form a three dimensional tissue-like structure but do not exhibit significant contractile forces to cause the forming cell-matrix construct to contract and peel itself from the culture substrate. Media exchanges are made every two to three days with fresh matrix production medium and with time, the secreted matrix increases in thickness and organization. The time necessary for creating a cell-matrix construct is dependent on the ability of the initial seeding density, the cell type, the age of the cell line, and the ability of the cell line to synthesize and secrete matrix.

When fully formed, the constructs of the invention have bulk thickness due to the fibrous matrix produced and organized by the cells; they are not ordinary confluent or overly confluent cell cultures where the cells may be loosely adherent to each other. The fibrous quality gives the constructs cohesive tissue-like properties unlike ordinary cultures because they resist physical damage, such as tearing or cracking, with routine handling in a clinical setting. In the fabrication of a cultured dermal construct, the cells will form an organized matrix around themselves on the cell culture surface preferably at least about 30 microns in thickness or more, more preferably between about 60 to about 120 microns thick across the surface of the membrane; however, thicknesses have been obtained in excess of 120 microns and are suitable for use in testing or clinical applications where such greater thicknesses are needed.

Optionally, mixed cell populations of two or more cell types may be cultured together during the formation of a tissue construct of the invention provided that at least one of the cell types used is capable of synthesizing extracellular matrix. The second cell type may be one needed to perform other tissue functions or to develop particular structural features of the tissue construct.

The production of the matrix in vitro in accordance with the present invention has shown to mimic several of the processes exhibited in production of matrix as well as repair of matrix in vivo.

The following examples are provided to better explain the practice of the present invention and should not be interpreted in any way to limit the scope of the present invention.

EXAMPLES

Example 1

As taught by the present invention extracellular matrix extracts are used for the induction of organ/tissue specific cell phenotypes from cultured bone marrow-derived mesenchymal stem cells in vitro. These extracts contain all the growth factors and cytokines present in the extracellular matrix necessary for specific organ differentiation.

Specifically the present example is based on the production and use of cartilage extracellular matrix extracts.

In order to induce the stem cells to differentiate and produce extracellular matrix the method of the present invention requires the addition of an extracellular matrix extract (including soluble factors) closely resembling the matrix of the tissue cells into which the stem cells should differentiate.

Such inducing extracts may be prepared in various ways. In particular they may be derived directly from the organ/tissue of interest, either from a human or an animal, which matrix closely resembles that of a human. Alternatively, the extracts may be made synthetically by blending commercially available growth factors, differentiation factor etc in order to produce an extract closely resembling the corresponding in vivo extracellular matrix.

In the present example the extracellular matrix extracts are organ-derived extracts on equine basis (supplied by Ossacur, Oberstenfeld, Germany). These organ-derived extracts are sterile acellular lyophilizates extracted from cartilage. In order to demonstrate the applicability of these extracts to induce differentiation of the stem cells and extracellular matrix changes in cell morphology, proliferation rate, expression of typical chondrogenic differentiation markers have been examined. Most importantly, the identification of SOX9 (Crombrugghe et al. 2001 and Goldring et al. 2006), a major nuclear transcription factor expressed in cells undergoing condensation and required for the expression of aggrecan cartilage-specific matrix protein (Lin et al. 2005), and the presence of glycosaminoglycans

(GAGs) by alcian blue and toluidine blue staining (Terry et al. 2000), in cultured BMMSC for 14 days with two different concentrations of joint and meniscus extracts. A. Sample collection and isolation of bone marrow-derived human mesenchymal stem cells:

Bone marrow stem cells are primitive cells present within the bone marrow where they account for approximately 1 per 100,000 of the nucleated cells. Patients (n = 4) requiring bone marrow aspiration for the evaluation of hematologic problems were recruited. The skin overlying the iliac crest was prepared and draped. Through a small stab incision, ~ 3ml of bone marrow was aspirated through a bone marrow needle into a 10 ml syringe containing

EDTA. Bone marrow aspirate was then subjected to hypoaque density gradient technique by centrifugation at 1400 rpm for 30 minutes at room temperature. Buffy coat, containing nucleated cells, was isolated and re-suspended in 10 ml culture medium compromising

Dulbecco's modified Eagles medium (1000mg glucose/L, L-glutamine, NaHCO and pyridoxine.HCI), with 1 % penicillin / streptomycin (Sigma-Aldhch Co Ltd, Irvine UK) and 10% fetal bovine serum (GIBCO-BRL) and centrifuged for 10 minutes at 900 rpm. Supernatant was the removed and the cell pallet was re-suspended in medium by 3 successive aspirations and expulsions through a 23-gauge needle attached to a 5 ml syringe. Cells were then plated in a 100 mm petri dish and incubated at 37⁰C in a 95% air and 5% carbon dioxide at 100% humidity. After 7-10 days in culture, cells adhere and start forming colonies. After reaching ~ 90% confluency, cells were the trypsinized and seeded into new culture vessels, a process known as passaging (in vitro cell propagation). Cells at passage 5 were used in all assays.

B. Sample preparation for assays

Cells at passage 5 were trypsinized then counted using trypan blue. Depending on culture

3 3 vessel size a specific number of cells was seeded: 60 x 10 and 15 x 10 BMMSC were

2 seeded into a 25 cm flask and 12-well plate, respectively. Each vessel was then filled with the appropriate amount of standard medium. After 24 hours of seeding, cells were treated either with standard medium (control group) or medium containing cartilage extract at two different concentrations, 16μg/ml and 36μg/ml. Cultures were replenished with fresh media (with or without extract) every 3 days. TGF-β1 recombinant protein (R&D Systems) was used at a concentration of 10ng/ml as a positive control for the differentiation of BMMSCs into chondrocytes. Cell supernatants were collected and stored at -80^QC. C. Cytotoxicity and proliferation assays

Toxicity of the extracts on the cultured cells was established using CytoTox 96 Non- Radioactive Cytotoxicity (Promega). This assay quantitatively measures the lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis. Released LDH in culture supernatants is measured with a coupled enzymatic assay, which results in the conversion of a tetrazolium salt into a red formazan product, the absorbance of which is measured at 492 nm. Results were obtained 6 hours post treatment.

Proliferation was conducted using CellTiter 96 Non-Radioactive (Promega). This assay is a MTT-based method which measures the ability of metabolically active cells to convert tetrazolium salt into a formazan product. The absorbance is recorded at 595nm. Briefly,

4 cells were counted and seeded at 10 densities in a 96-well-round bottom plate. Cell proliferation was measured on days 1 , 3, 7 and 14. 100 μl was seeded into each well (triplicates). For background measurements, three wells containing media alone were seeded. 15 μl of dye solution was added into all wells. Cells were then incubated for 4 hours at 37^Q C, humidity 100%, 5%CO . Two 10Oμl aliquots were then removed from each well and transferred to a 96 well plate. 100 μl of stop solution were then added into all wells. Cells were then incubated for minimum 1 hour at 37⁹ C, humidity 100%, 5%CO .

Absorbance was measured at 595nm.

D. Light microscopy for morphological changes

On day 1 , 3, 7 and 14 of cultures, morphology of the cells was observed using DIC light microscopy. Changes in morphology were documented using digital photography. Photographs were taken at different time points post treatment.

E. Characterization of bone marrow mesenchymal stem cells

The presence of Stro-1 cell surface marker was used to confirm that cell cultures comprised a multipotential bone marrow stem cell population. Cells expressing the Stro-1 cell surface

4 marker were identified using immunofluorescent method. 1.5 x10 bone marrow stromal stem cells were seeded in a 12-well plate onto sterile cover slips. One coverslip with cells was then placed into each well of a 12 well culture plate. Wells were supplemented with control medium containing Dulbecco's modified Eagles medium (1000mg glucose/L, L- glutamine, NaHCO3 and pyridoxine.HCL, Sigma Aldrich D6046), with 1 % penicillin /streptomycin and 10% bovine fetal bovine serum (FBS) (Gibco BRL). At 36 hours cells were fixed with 70% ethanol and then washed 3 times with phosphate buffer saline (PBS) (Cambrex). Cells were then incubated with Stro-1 monoclonal antibody (R&D systems, Minneapolis, MN) at 10 μg/ml at 37^SC for 1 hour. Primary antibody was then removed from the wells and cells were washed three times with phosphate buffered saline, before incubation for 1 hour with anti mouse IgM FITC-conjugated (Molecular Probes) at a concentration of 1 :1000 for 1 hour . Secondary antibody was removed and cells were further washed 3 times with PBS. A negative control was performed with the secondary antibody. After the incubation period cells were washed, mounted onto slides and observed using a fluorescent microscope.

F. Histological Staining

1. Hematoxylin and Eosin (H&E) H&E stain was used to assess both the morphology of the cells and the presence of calcium deposits in the culture. 14 days after treatment, cells seeded on coverslips were fixed with 70% ethanol, hydrated with distilled water for 3 minutes, placed in hematoxylin for 4 minutes then washed once with tap water for 3 minutes. Next, the coverslips were dipped in eosin for 45 seconds followed by a dehydration step consisting of 95% ethanol for 3 minutes (twice) then 100% ethanol for 3 minutes. Cells were then placed in xylol for 3 minutes then mounted. Cells were visualized using light microscopy.

2. Toluidine Blue

Toluidine blue is a monocationic dye which has been used for the staining of proteoglycans (PGs) in tissue for histochemical studies. Cells were hydrated using water then incubated 2-

3 minutes in toluidine blue stain (pH 2). Three consecutive washes with distilled water were performed after which the cells were dehydrated using 95% and then 100% ethanol (10 quick dips to avoid fading of stain). Cover slips were then cleared twice in xylene for 3 minutes each step. Coverslips were mounted and observed using light microscopy.

3. Immunofluorescence

3

Cells were seeded in a 12-well plate at a density of 15 x 10 . 14 days post treatment; wells were washed once with phosphate buffered saline (PBS) and then fixed with 70% ice-cold ethanol over night. Cells were then blocked in 3% normal goat serum (NGS) for 1 hour after which primary antibody (mouse anti-aggrecan antibody (Chemicon, USA)) was applied at a concentration of 1 μg/ml and incubated over night at 4³C. Primary antibody was then removed from the wells and cells were further given 3 washes with phosphate buffered saline followed by incubation for 1 hour with a secondary antibody (anti mouse IgM FITC- conjugated (Molecular probes, USA)) at a concentration of 1 :1000 for 1 hour . Secondary antibody was removed and cells were further washed 3 times with PBS. A negative control was performed with the secondary antibody. After the incubation period cells were washed, mounted onto slides using antifade gold and observed using a fluorescent microscope.

G. Quantification of Chondroitin Sulfate Alcian blue is a tetracationic dye which has been used by various investigators for the quantification of microgram quantities of nonradioactive GAG and PG. GAGs and PGs were precipitated specifically by addition of Alcian blue under conditions of low pH and of critical electrolyte concentrations, which prevent interaction of the dye with other anionic macromolecules such as nucleic acids or proteins. Cells were washed with PBS then incubated in Alcian Blue stain (1% Alcian Blue in 3% acetic acid) for 20 min. Cells were destained by three rinses in 3% acetic acid and then washed once in water. To quantify stain incorporated, cells were solubilized in 1 % SDS by shaking for 30 min at room temperature and then heating to 90 ⁰C for 1 h. Absorbance was detected at 595 nm.

H. lmmunoprecipitation of TGF β1

20 μl of agarose A beads were added to 100 μl of joint and meniscus bovine extract and left to rotate at 4⁰C for 2 hours. This is a pre-clearing step to remove excess IgG. Samples were then centrifuged for 10 minutes at 12500 rpm. Supernatant was transferred to a new tube and 1 μg of TGF-β1 antibody was added. Samples were left to rotate for 1 hour at 4⁰C. After 1 hour, 20 μl of agarose A beads were added and left to rotate over night at 4O. The next day, samples were centrifuges for 2 minutes at 12500 rpm. The pellet was washed three times with phosphate buffered saline (PBS), and then centrifuged for 2 minutes at 12500 rpm. The supernatant was removed and the pellet was re-suspend in 40 μl 2X sample buffer 5% β-mercaptoethanol. The samples were boiled for 5 minutes and ran on SDS-PAGE gel. Note: samples were processed in parallel but without the addition of TGF-β1 antibody.

I. Western Blot Analysis

Cells were washed once with phosphate buffered saline (PBS) then solubilized in lysis buffer consisting of 0.125M Tris-HCI (pH 6.8), 2% SDS, 5 % B-mercaptoethanol, and 10% glycerol. Samples were loaded onto a 12% SDS-polyacrylamide gel, subjected to electrophoresis, and transferred to PVDF membrane. After blocking the membranes in 5% skim milk in Tris-buffered saline containing 0.05% Tween-20, the blots were incubated with specific antibodies (anti-TGFβ1 antibody). The blots were washed, and protein bands were visualized by chemiluminescence. Proteins were coomassie stained to ensure both equal loading and quality of the proteins extracted. GAPDH was also used for equal loading.

J. Osteopontin secretion

Osteopontin (OPN) secretion was analyzed using a human OPN TiterZyme enzyme-linked immunosorbent assay (ELISA) Kit (Assay designs). Cell supernatant was collected at day 14 post treatment. A volume of 100 μl assay buffer provided in the kit and 100 μl of the samples or standard solution were added into a microtiter plate. A series of standard solutions were prepared by diluting the stock solution containing recombinant human OPN (16ng/ml). The micotiter plate with the samples was incubated at room temperature for 1 h, the solutions in the wells were aspirated and the wells were rinsed by adding 400μl of washing buffer 4 times. Then 100 μl of the labeled antibody solution was added to each well and the plate was sealed and incubated for 30 minutes at room temperature. The antibody solution was aspirated and the wells were rinsed by adding 400μl of washing buffer 4 times. Subsequently, a volume of 100 μl of substrate solution was added to each well and incubated for 30 minutes at room temperature. The reaction was stopped by the addition of trisodium phosphate to each well. The absorbance of each well at 405 nm was measured using a microplate reader.

K. RNA extraction and Real Time PCR

Total cellular RNA was extracted using NucleoSpin RNA Il from Macherey-Nagel. We used real-time PCR primers that recognize Collagen type I (NM000088), SOX9 (NM_000346),

Aggrecan (NM 013227) and GAPDH. Amplification of the house keeping gene GAPDH was used to verify RNA quality and ensure equal loading of the samples. Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed using LightCycler (Roche

Diagnostic). The real-time PCR conditions consisted of a reverse transcription step for 20 minutes at 61⁹C to generate single-stranded cDNA from mRNA followed by a reverse transcriptase inactivation and initial denaturation for 30 seconds at 95⁹C. This was followed by 40 cycles of amplification consisting of 95⁹C for 1 second, 58⁹C for 15 seconds and 72⁹C for 13 seconds. A final cooling step at 40⁹C for 30 seconds was then performed. The concentration of GAPDH was used to control for input of RNA and to normalize all genes tested from the same cDNA sample. RESULTS

A. BMMSC isolation, characterization and morphology Approximately 2.5 - 4 ml of bone marrow aspirates were obtained from patients after obtaining their informed consent. After ficoll, the yield of nucleated cells extracted was 1 10x10⁴ cells per 1 ml of bone marrow aspirate. Cells were then plated, replenished with new culture medium every 3 days. Cultured BMMSC were observed by light microscopy and morphology was documented using digital photography. After 7-10 days in culture, cells started to adhere to the plastic of the culture vessel forming colonies. At day 14, these cells were trypsinized and seeded into new culture vessels, a process known as passaging. With repeated passaging, the cells exhibited a characteristic elongated morphology. The identity of these cells as BMMSC was conformed by staining for Stro-1 , an antigen marker specific for BMMSC. These cells positively stained for Stro-1 .

B. Effect of organ derived matrix extracts on cellular cytotoxicity and proliferation Cytotoxicity assay was performed to ensure the non-cytotoxic effect of extract on the cells. Results showed negative cytotoxic effect on BMMSC (Fig 1 A). In parallel to the cytotoxicity assay, a proliferation assay was performed to test the growth/proliferation rate of BMMSC in the presence of two different concentrations (16μg/ml and 32μg/ml) of cartilage derived extracts. Cellular proliferation was evaluated at different days in culture (day 1 , 3, 7, and 14) (Fig 1 B). Cellular proliferation was found to be reduced in cells treated with the extracts as compared with control untreated cells suggesting cell differentiation. In non-treated cells, proliferation increased significantly. BMMSC treated with meniscus decreased the proliferation rate more than those treated with joint extract.

C. Morphological changes post treatment with organ specific matrix extracts

On day 1 of culture, BMMSCs in control and extract containing medium showed spindle phenotype typical of bone marrow-derived stem cells. Treatment with both cartilage (joint and meniscus) matrix extracts, at two different concentrations 16μg/ml and 32μg/ml, induced prominent morphological changes starting day 7 when treated with joint and meniscus. It was noted that medium alone did not induce any changes. Seven days post treatment with joint and meniscus; cell aggregates were noticed with a central alignment containing pre-hypertrophic cells in the middle indicative of chondrocytic differentiation (Fig 6). Control cultures, studied at all time points, maintained a spindle shaped morphology. D. Identification of TGF-β1 in cartilage derived extracts

TGF-β1 is one of the growth factors known in the enhancement of chondrocytic differentiation of BMMSC. TGF-β1 was identified in of joint and meniscus matrix extracts using an immunoprecipitation technique.

E. Expression of chondro-specific differentiation markers

Several specific markers have been identified in the differentiation of BMMSC along chondrocytic lineage. The most commonly used markers in chondrocytic differentiation are Sox9, aggrecan and GAG production.

a. Sox9

Chondrogenesis has been shown to be driven by a series of transcription factors (Erlebacher 1995 and de Crombrugghe 2000) most importantly Sox9. Sox9 is necessary for chondrogenic differentiation both before and after mesenchymal condensations (Toshiyuki

2005). Sox9, being a key transcription factor that plays a role in chondrogenesis, was significantly increased upon the treatment with cartilage derived matrix extracts (joint and meniscus) as compared to control untreated BMMSC at day 14 post treatment. Joint and meniscus extracts induce chondrogenic differentiation of BMMSCs through the upregulation of Sox9 mRNA (Fig 2). Whereas, on the other hand, Collagen type I, known to be upregulated during osteogenic differentiation, was not detected (Fig 3). This data suggests the onset of chondrogenesis.

b. Aggrecan Aggrecan, a large chondroitin sulfate proteoglycan, is one of the major structural components in cartilage matrix. It binds to hyaluronan and Link proteins to form huge aggregates. The aggrecan molecule is composed of a core protein and glycosaminoglycans, mostly chondroitin sulfate. Aggrecan was detectable at the protein level at day 14 post treatment using mouse anti-aggrecan antibody. The expression of aggrecan was highly distributed and localized in chondrocytic differentiating human mesenchymal stem cells as compared to untreated cells (Fig 4). In parallel, staining with the secondary antibody was performed to ensure the specific binding of anti-aggrecan antibody (data not shown). Aggrecan positive cells were also counted proving BMMSCs treated with bovine joint and bovine meniscus extract, and in parallel BMMSCs treated with TGF-β1 recombinant protein alone, were more in number as compared to the control untreated sample (Fig 5). However, at the transcriptional level, aggrecan mRNA was decreased as compared to untreated cells but significantly increased in TGF-β1 treatment. This might suggest that at day 14, aggrecan expression was translated from mRNA to protein.

c. Determination of αlvcosaminoαlvcans (GAGs)

Alcian blue and Toluidine blue dyes form complexes with anionic glycoconjugates (AG) such as proteoglycans (PG) and glycosaminoglycans (GAG). In our assay, alcian blue was used to quantitatively measure the amount of chondroitin sulfate in cell lysates. Our results showed an increase in the amount of chondroitin sulfate in both joint and meniscus extracts as compared to the control untreated cells. It was noted that cells treated with bovine joint extract contained more chondroitin sulfate than that of bovine meniscus extract. The addition of 10ng/ml TGF-β1 to the culture was conducted in parallel manifesting an increase in the level of chondroitin sulfate. In parallel, toluidine blue staining is used to histologically detect glycosaminoglycans (GAGs) in BMMSC cultures in the presence of joint and meniscus extract. Results show a clear blue cytoplasm with violet/red purple granules indicative of the presence of glycosaminoglycans GAGs.

F. Cartilage derived matrix extracts do not induce osteocytic differentiation of BMMSC in vitro

a. Osteopontin (OPN) secretion

Osteopontin is known to be secreted and expressed as a result of BMMSC differentiation into osteocytes and not chondrocytes. In order to detect osteopontin (OPN) secretion, cellular supernatant was collected from all cultures. Levels of OPN were same as that of the control untreated cells. Colloss and Colloss E, previously used in differentiating BMMSC into osteocytes (El-Sabban et al. 2007), treated cultures showed high levels of secreted OPN in their supernatant. For this assay, 10ng/ml of BMP-2 and BMP-7 (OP-1 ) recombinant proteins were used as a positive control for the induction of BMMSC differentiation into osteocytes. In these cultures, osteopontin was also secreted. In contrast, cartilage derived joint and meniscus extracts did not induce the secretion of osteopontin into the supernatant.

b. Collagen type I mRNA expression

Treatement of BMMSC with cartilage derived matrix extracts (joint and meniscus) resulted in low levels of collagen type I equivalent to that of control untreated cells. On the other hand, a significant increase in collagen type I expression was detected in BMMSC treated with bone derived matrix extracts (Colloss and Colloss-E) as compared to untreated BMMSC. This suggests the exclusive expression of collagen type I in BMSC differentiation into osteocytes rather than chondrocytes.

c. Calcium deposits

A number of studies have proven the deposition of calcium as a marker of osteocytic differentiation and maturation (Wang 1999, Salasznyk 2004 and Heungsoo 2005). Thus, mineralization is a crucial indicator of osteogenesis and osteogenic differentiation and not chondrocyte differentiation (Sikavitsas 2002, Pittenger 1999 and Jaiswal 1997). Cells treated with Colloss, Colloss E, joint and meniscus extract were stained with hematoxylin and eosin (H & E) and analyzed using light microscopy. Cells treated with Colloss and Colloss E became flattened and polygonal with calcium dots near the nucleus indicating osteocytic differentiation of these cells. In contrast, cells treated with cartilage joint and meniscus became rounded in shape an indication of hypertrophy of the cells and absence of calcium deposits, thus indicating chondrocytic differentiation of BMMSCs.

Control untreated cells maintained their spindle shape.

G. Extra cellular matrix extract obtained from differentiated stem cells.

After 14 days of cultivation (cf section A and Figure 1 B) the cultivation broth, comprising differentiated human cells and extracellular matrix, was centrifuged. The supernatant constitutes the extracellular matrix extract of the present invention. This human based extract has a composition which more closely resembles the extracellular matrix of human cartilage tissue than the equine base extract does. Accordingly, the present invention provides a more perfectly resembling extracellular matrix extract than the prior art envisages.

Example 2

In addition to extracellular matrix resembling those derived from animals (or humans if available) for the stimulation of stem cells to produce extracellular matrix of a desired tissue the present invention also contemplates more simple extracellular extracts resembling those derived from animals or humans; instead of deriving the extract directly from the tissue of interest it is possible to manufacture a more simple but still useful extract for the stimulation of stem cells to produce extracellular matrix. The present inventors have been able to stimulate stem cells to produce extracellular matrix of bone, cartilage, bone marrow, pancreas and liver by simply mixing human growth and differentiation factors in appropriate ratios in accordance with Table 1.

Table 1

Example 3

Bone marrow-derived mesenchymal stem cells (BMMSCs) are infrequent multipotent cells that can differentiate into multiple lineages, such as bone, cartilage and muscle cells among others. Hence, these cells are of potential clinical importance for the repair of damaged tissues. The local microenvironment in vivo is critical to support the desired differentiation of stem cells or to sustain the phenotype of the stem cell-derived in vitro differentiated cells. This local microenvironment comprises a physical support supplied by the organ matrix as well as tissue-specific cytokines. Bone matrix contains collagen type 1 and many of the growth factors involved in the cascade of bone formation. Consequently, when bone marrow is used to enrich orthopedic grafting matrices, it almost invariably produces faster and more consistent defect healing compared with bone marrow or the carrier matrix alone.

In this Example, the effect of Colloss and Colloss-E on adult in vitro expanded BMMSCs has been assessed. Specifically, it is demonstrated that these xenogenic bone matrix extracts (BMEs) induces osteoblastic differentiation of cultured BMMSCs, which means that xenogenic bone matrix extracts can be used to differentiate human stem cells into the desired tissue, whereby a human derived extracellular matrix and soluble factors are produced, which can then be isolated for later differentiation of human stem cells into the osteoblasts.

Materials and Methods

Preparation of Colloss^® & Colloss-E^®

Bovine and equine bone protein extracts, Colloss and Colloss-E, respectively, were supplied by Ossacur, AG (Oberstenfeld, Germany). These extracts were prepared, aseptically, from cortical diaphysis of long bones from disease-free, young (<12 months old) calves and foals of closed herds. Briefly, bones were pulverized and delipidated with acetone for 60 min at 4"C. The resulting bone particles were demineralized in 0.6N hydrochloric acid for 60 min at 4⁰C. Particles were then washed in deionized water, extracted with 4M guanidine hydrochloride and ultrafiltered using 3 K nominal molecular weight cut-off membranes. Colloss and Colloss-E are normally supplied as sterile lyophilizates that comprise a suspension when reconstituted. Hence, Colloss and Colloss-E dispersion in water, an intermediate stage from the manufacturing procedure, was used for this study.

Cells, treatment & antibodies

Six different patients requiring bone marrow aspiration for routine follow-up of hematologic disorders, who had signed the informed consent, were recruited. All used bone marrow samples that were used exhibited normal morphology. This study was approved by the institutional review board (IRB) of the American University of Beirut, Lebanon.

Human BMMSCs were isolated from 3^ ml of bone marrow aspirates using Ficoll density gradient (Ficoll-Paque™ Plus, Amersham Bioscience, UK). Cells were cultured in Dulbecco's modified Eagles medium (1000 mg/l-glucose/L, L-glutamine and sodium pyruvate; Sigma Aldrich, USA), with 1 % penicllin/streptomycin, fetal bovine serum (Gibco BRL, USA) at 37⁰C in 95% air with 5% carbon dioxide at 100% humidity. Medium was replenished every 2 days. Confluent cultures were passaged by trypsinization and gentle scraping. Cells between passage five and seven were used for all experiments.

For the assessment of osteoblastic differentiation, 1.5 x 10⁴ or 3 x 10⁴ BMMSCs were seeded in 12- and six-well plates, respectively, and treated with 1 :50 dilution of either Colloss or Colloss-E (28 and 32 μg/ml, respectively). Morphological changes were documented by photo-microscopy on a daily basis. Cell proliferation was assessed using the CellTiter 96^® (Promega, USA). Expression of Stro-1 and osteopontin was evaluated by fluorescence immunocytochemistry using specific antibodies (R&D systems, USA).

Alkaline phosphatase activity

BMMSCs were treated for 7 or 14 days with medium containing 1 :50 Colloss or Colloss-E (28 and 32 μg/ml, respectively). Alkaline phosphatase protein levels in total cell lysates were evaluated by western blot using specific antibodies (Abeam, UK). Alkaline phosphatase enzyme activity was assessed in medium-free cell lysates as described by Oreffo and colleagues using 5 mM of p-nitrophenyl phosphate (Amresco, USA) as a substrate in 2- amino-2-methyl-1 propanol (Sigma) alkaline buffer solution.

Osteopontin secretion

BMMSCs were treated for 7, 14 and 30 days with medium containing Colloss or Colloss-E

(28 and 32 μg/ml, respectively). Osteopontin levels were determined in cell-free supernatant using a human OPN TiterZyme ELISA kit (Assay designs, USA) as recommended by the manufacturer.

Calcium deposits

BMMSCs were treated for 15 days with medium containing 1 :50 Colloss or Colloss-E (28 and 32 μg/ml, respectively). Cultures were stained with hematoxylin and eosin (H&E) and calcium deposits were counted.

ELISA

Protein levels of various osteogenic cytokines in Colloss-E were determined by ELISA (antibodies generated against human factors), according to the supplier's instructions. Levels of BMP-2, TFG-βi and insulin-like growth factor (IGF)-I were measured using commercial ELISA kits from R&D Systems, whereas levels of BMP-7 were measured using commercial ELISA kits from RayBiotech.

Statistics

Statistical analysis was determined by analysis of varience (ANOVA) using SPSS for windows. The results are expressed as mean ± standard error, p-values of 0.05 were considered significant.

Bovine & equine BMEs induce morphological changes of BMMSC

BMMSC cultured at passage six exhibited a characteristic elongated morphology (Figure 7A) and expressed the cell surface marker Stro-1 (data not shown), confirming the presence of bone marrow stromal stem cells as described. Treatment with 1 :50 and 1 :100 dilution of either Colloss or Colloss-E, but not with medium alone, induced characteristic morphological changes, starting at day 3 and becoming more obvious at day 7. In general, cells became flattened and exhibited cytoplasmic projections suggestive of osteoblastic differentiation (Figure 7A and not shown). This was accompanied by a marked decrease in total cell density (Figure 7B) and cell proliferation (Figure 7C).

Bovine & equine BMEs induce osteoblastic differentiation of BMMSCs

To evaluate whether Colloss or Colloss-E induce osteoblastic differentiation of BMMSCs, treated cells were assessed for the presence of osteoblast-specific markers, such as alkaline phosphatase activity, osteopontin secretion and calcium deposits. Both Colloss and Colloss-E induced a sharp increase in alkaline phosphatase protein level at day 14 (Figure 8A). This was accompanied by a significant increase in alkaline phosphatase enzyme activity in medium-free cell extracts at day 14 (Figure 8B). Of note, Colloss and Colloss-E were devoid of alkaline phosphatase activity (data not shown). Furthermore,

Colloss and Colloss-E upregulated both cellular (Figure 9A) and secreted (Figure 9B) osteopontin expression (p < 0.01 for Colloss, p < 0.05 for Colloss-E). Most importantly, while untreated BMMSCs did not show any evidence of calcium deposits as detected by H&E stain, both Colloss- and Colloss-E-treated cells exhibited a significant number of calcium deposits (Figure 10A & B) (p < 0.05 for Colloss and Colloss-E). Altogether, these results strongly suggest that both Colloss and Colloss-E induce osteoblastic differentiation of in vitro expanded BMMSCs.

Colloss-E^® contains osteogenic growth factors

The fact that Colloss and Colloss-E induce osteoblastic differentiation of in vitro expanded BMMSCs suggests that these BMEs contain osteogenic growth factors, as reported for

DBM. Indeed, ELISA assays revealed that Colloss-E contains significant amounts of BMP-2 (2.6 ± 0.2 mg/g), BMP-7 (3.8 ± 2.7 mg/g), TGF-β1 (55 ± 1 1 mg/g) and IGF-1 (2.9 ± 0.8 mg/g).

Discussion

In this Example it has been demonstrated that both xenogenic BMEs induce osteoblastic differentiation of in w^'fro-expanded BMMSCs. These findings have been corroborated with in vitro studies demonstrating the osteoinductive effect of DBM. Indeed, human DBM induced osteoblastic differentiation of a murine muscle-derived cell line and, more importantly, of human bone marrow stromal cells. Similarly, demineralized rabbit bone extracts increased the rate of osteoblastic differentiation of murine fibroblasts. Furthermore, bovine bone extracts induced osteoblastic differentiation of rat mesenchymal cells with increased alkaline phosphatase activity and decreased proliferation. A recent study explored the effects of Colloss on the human mesenchymal stem cell line that overexpresses human telomerase reverse transcriptase (TERT; hMSC-TERT), which maintains in vitro proliferation and osteoblastic differentiation potential. In this study, Colloss had opposite effects depending on the differentiated state of the cells. Whereas, Colloss increased proliferation and decreased alkaline phosphatase activity of undifferentiated hMSC-TERT cells, it diminished proliferation and increased alkaline phosphatase activity and collagen synthesis of osteoblastic differentiated hMSC-TERT cells. However, our report is the first to demonstrate that Colloss and Colloss-E induce osteoblastic differentiation of in wf/O-expanded human BMMSCs.

The mechanism of xenogenic BME-induced osteoblastic differentiation of BMMSCs remains to be understood. Normal in vivo cell differentiation is coordinated by both a structural scaffold and soluble cytokines, both provided by the microenvironment of target organs. The use of organ-specific extracts to supply stem cells with a milieu that mimics the in vivo microenvironment is an attractive alternative to the use of a defined combination of cytokines and growth factors. These extracts provide both the physical and biochemical signals in relative physiologic proportions. Indeed, xenogenic BMEs consist of collagens as well as noncollagenous-associated proteins. Here, we show that Colloss-E contains significant amounts of osteogenic growth factors, such as BMP-2, BMP-7, TGF-β1 and IGF- 1. Since the ELISA kits were optimized for human proteins, it is difficult to accurately assess the exact levels of growth factors in our extracts. Furthermore, since these cytokines were previously reported to be osteogenic, their presence in Colloss-E probably contributes to the osteoblastic differentiation of BMMSCs. BMPs induce osteoblast differentiation from nonosseous cells in vitro and ectopic bone formation in vivo, which suggest a direct effect of BMPs on stem cells. Similarly, Colloss-E enclosed in titanium mesh and implanted subcutaneously in rats induced bone formation. However, although the role of collagen alone in osteoblastic differentiation in vitro, or osteoinduction in vivo, remains controversial, the collagen component in Colloss and Colloss-E could play an important role in attracting mesenchymal stem cells to the area of bone defect and in allowing exposure of stem cells to growth factors.

Finally, although the mechanism remains unknown, both Colloss and Colloss-E induce osteoblastic differentiation of in vitro expanded BMMSCs, which has far-reaching applications in the treatment of bone defects. These extracts can be co-administered with autologous BMMSC in the area of the bone defect, to enhance their osteoblastic differentiation. Alternatively, Colloss and Colloss-E can be used as an ex vivo tissue engineering scaffold to induce in vitro osteoblastic differentiation of expanded BMMSCs, which can then be applied in vivo. This provides a safe, inexpensive and readily available source of osteoinductive graft materials. Example 4

The present example illustrates the applicability of the present invention to differentiate human BMMSCs into hepatocytes thereby producing valuable human based ECM and soluble factors. Upon differentiation of the BMMSCs into hepatocytes the ECM and soluble factors are obtained by removing the differentiated cells leaving ECM and soluble factors; commonly denoted ECM extract. This extract may then be stored and later used as differentiation medium for the differentiation of BMMSCs into hepatocytes (ex vivo or in vivo). The differentiation of the human BMMSCs into hepatocytes is visualized in Figure 13, whereas the biochemical changes associated therewith are shown in Figures 1 1 and 12.

BMMSC isolation and culture: Human BMMSC were isolated from 3-4ml of bone marrow aspirates using Ficoll density gradient. Cells were cultured in DMEM (1 g/L glucose) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum at 37C in 95% air with 5% carbon dioxide at 100% humidity. Medium was replenished every 3 days. Confluent cultures were passaged by trypsinization and gentle scraping. Cells at passage 5 were used in the experiments.

Human hepatocvte cell culture:

Human hepatocytes were cultured in DMEM (1g/L glucose) supplemented with 1 % non essential amino acids, 1% penicillin/streptomycin, 1 % glutamax and 10% fetal bovien serum at 37C in 95% air with 5% carbon dioxide at 100% humidity. Cells were cultured for a maximum of 15 days after which new cells were put in culture.

BMMSC differentiation

Human BMMSCs were seeded in 6-well format inserts at a density of 30000 cells. In the compartment underneath the inserts, human liver cells were seeded at the same density. Cells were replenished with fresh growth media every 3 days. The media used to replenish the cells was each cell growth media. Cells were harvested 10 days post co-culture. Human liver cells growth media: DMEM low glucose, non essential amino acids, fetal bovine serum, glutamax, penicillin/streptomycine.

BMMSC growth media: DMEM low glucose, fetal bovine serum, peniciline/streptomycine.

Preparation of ECM and conditioned media

Human liver cells were cultured in T75 flasks. After 48h of cell counfluency, the media was removed and filtered using 0.2um syringe filter and stored at -80C. The liver cells were then washed once after which a "removing reagent" was added to the cells ensuring total removal of cells while leaving the deposited ECM intact. The ECM was gently washed once with media (without FBS) to remove any traces of the removing agent and then the ECM was scraped in 2 ml of media (without FBS) and aliquoted and stored at -80C.

Differentiation of BMMSC into hepatocvtes by culturinq on human liver ECM In order to test whether or not the liver ECM extract obtained in accordance with the above protocol cell culture flasks coated with human liver ECM extracts were seeded with BMMSC. Cells were allowed to interact with matrix for 10 days, RNA is extracted and hepatocyte differentiation markers monitored.

As shown in Figure 1 1 hepatocyte markers induced in BMMSC: 1 - human hepatocyte cells express the various liver markers. 2- On the other hand, undifferentiated BMMSC do not express liver markers. 3- When BMMSC differentiate into hepatocytes these cells start expressing liver specific markers: AFP and p450.

As can be derived from Figure 12 the induction of p450 expression in ECM differentiated

BMMSCs takes place, which verifies that the alleged differentiation has taken place. Undifferentiated BMMSCs do not express p450 (1 ). After culturing BMMSCs on human liver cell ECM, p450 expression was induced (2) and increased induction was detected upon the treatment with 1 mM phenobarbital for 1 h (3) and 4h(4). House keeping gene was used to ensure equal loading of the samples.

Figure 13 shows the morphology of differentiated BMMSCs cultivated on human liver cell ECM. BMMSCs cultured on plastic maintained their spindle shape morphology (right panel), where as when cultured on human liver ECM for 10 days these cells changed to round shaped cells (left panel). In Figure 14 the expression of Liver-specific connexins in differentiated stem cells (hepatocytes) can be seen. This indicates that the liver tissue is being generated.

Figure 15 confirms that the liver-differentiated BMMSC indeed work as hepatocytes with the specific induction of p450.

In Figure 16 the glucose uptake in liver-differentiated BMMSC has been compared to undifferentiated BMMSC (hepatocytes). Clearly differentiated cells have a much higher influx of glucose indicating that they have the glucose storage potential known from native hepatocytes.

Claims

1. A method for producing a human-based extracellular matrix extract and soluble factors of a desired human tissue, comprising,

• seeding human stem cells in a culture vessel in an environment comprising extracellular matrix resembling the extracellular matrix of the desired tissue,

• culturing the cells in order to differentiate them into cells of the desired tissue, whereby the cells are stimulated to synthesize, secrete and organize extracellular matrix and soluble factors; • continued culturing of the cells until the cells have been differentiated into cells of the desired tissue and have synthesized extracellular matrix and soluble factors, and

• removing the cells to obtain the extracellular matrix extract and soluble factors of the desired tissue.

2. The method of claim 1 , wherein the extracellular matrix resembling the extracellular matrix of the desired tissue is of animal origin or human origin.

3. The method of claim 1 or 2, wherein the extracellular matrix resembling the extracellular matrix of the desired tissue contains no non-human components.

4. The method of any one of the claims 1 to 3, wherein the extracellular matrix resembling the extracellular matrix of the desired tissue has been obtained my mixing human growth and differentiation factors known to play a role in the differentiation of the stem cells into the desired tissue.

5. The method of any one the claims 1 to 4, wherein the desired tissue is selected from the group consisting of liver, pancreas, cartilage, and bone-marrow.

6. The method of any one the claims 1 to 5, wherein the cells are removed by centrifuging the differentiated cells.

7. The method of claim 6, wherein the cells are genetically modified to produce a growth factor, hormone, peptide, or protein.

8. A human-based extracellular matrix extract and soluble factors obtainable by the method according to any one of the claims 1 to 7.

9. Method for differentiating bone human marrow-derived stem cells into chondrocytes, comprising:

• providing bone marrow-derived stem cells from a human;

• treating the stem cells with an extract and soluble factors of claim 8, wherein the desired tissue is cartilage tissue; and

10. Method for differentiating bone human marrow-derived stem cells into hepatocytes, comprising:

• providing bone marrow-derived stem cells from a human;

• treating the stem cells with an extract and soluble factors of claim 8, wherein the desired tissue is liver tissue; and

• cultivating and differentiating the stem cells until a sufficient amount of hepatocytes have been obtained.

11 . Method for differentiating bone human marrow-derived stem cells into beta- cells, comprising:

• providing bone marrow-derived stem cells from a human;

• treating the stem cells with an extract and soluble factors of claim 8, wherein the desired tissue is pancreas tissue; and

• cultivating and differentiating the stem cells until a sufficient amount of beta- cells have been obtained.

12. Use of extracts according to claim 8 for differentiation of stem cells.

13. Use according to claim 12 for differentiation of stem cells into at least one type of cells selected from the group consisting of chondrocytes, hepatocytes and beta- cells.