WO2007122233A1

WO2007122233A1 - Preparation of mesenchymal progenitor cells, particularly osteogenic progenitor cells

Info

Publication number: WO2007122233A1
Application number: PCT/EP2007/053991
Authority: WO
Inventors: Ileana Mateizel; Karen Sermon; Inge Liebaers; André VAN STEIRTEGHEM
Original assignee: Vrije Universiteit Brussel
Priority date: 2006-04-25
Filing date: 2007-04-24
Publication date: 2007-11-01

Abstract

The invention relates to in vitro methods for differentiation of primate pluripotent stem cells into mesenchymal progenitor and/or stem cells and to further uses of so differentiated cells. It provides a simple and reliable method to effect such differentiation.

Description

PREPARATION OF MESENCHYMAL PROGENITOR CELLS, PARTICULARLY

OSTEOGENIC PROGENITOR CELLS

Field of the Invention

The invention relates to in vitro methods for differentiation of primate pluripotent stem cells into mesenchymal progenitor or like cells, preferably mesenchymal progenitors capable of and/or committed towards differentiation along the osteogenic pathway, and to further uses of so differentiated cells.

Background to the Invention

Recent discoveries have raised expectations that stem cells may be a source of replacement cells and tissues that are damaged in the course of disease, infection, or because of congenital abnormalities. Various types of putative stem cells differentiate when they divide, maturing into cells that can carry out the unique functions of particular tissues, such as the heart, liver, brain, muscle or connective tissue.

A particularly important discovery has been the isolation and culture of pluripotent stem (PS) cells, which are thought to have the potential to differentiate into any cell type. The next challenge in developing the technology is to obtain dependable conditions for driving differentiation towards particular cell lineages that are desired for therapeutic purposes.

Early work on embryonic stem cells was done in mice (reviewed in Robertson. 1997. Meth Cell Biol 75: 173; Pedersen. 1994. Reprod Fertil Dev 6: 543). Pluripotent stem cells from primates were first successfully isolated and propagated by Thomson et al. (US 5,843,780; Proc Natl Acad Sci USA 92: 7844, 1995). These authors subsequently derived human embryonic stem (hES) cell lines from human blastocysts (Science 282: 1145, 1998). Gearhart and colleagues derived human embryonic germ (hEG) cell lines from foetal gonadal tissue (US 6,090,622; Shamblott et al. 1998. Proc Natl Acad Sci USA 95: 13726). Both hES and hEG cells have characteristics of pluripotent stem (PS) cells: they can be cultured extensively without differentiating, they retain a normal karyotype, and they are able to differentiate into a number of important cell types originating from the three embryonic germ layers - ectoderm, mesoderm and endoderm.

Efforts to differentiate primate, esp. human, pluripotent stem cells in vitro typically involve the formation of cell aggregates, either by overgrowth of the pluripotent stem cells cultured on feeder layers, or by forming embryoid bodies in suspension culture. The embryoid bodies generate cell populations with a highly heterogeneous mixture of phenotypes representing a spectrum of different cell lineages, including a number of ectodermal, mesodermal, and endodermal derivatives.

It is not straightforward to control and standardise differentiation using aggregates of pluripotent stem cells, since the aggregates or embryoid bodies contain multiple cell lineages and their composition may often depend on parameters, such as the aggregate size, which can be difficult to control. In addition, the step of producing the aggregates or embryoid bodies requires specific handling of the pluripotent stem cells and hence entails expenditure of time and labour and a potential risk of contamination. Production of multipotent stem cells, committed precursor cells or fully differentiated cells from primate, esp. human, pluripotent stem cells can therefore benefit from differentiation protocols which do not involve producing cell aggregates or embryoid bodies. Such derivatives can be particularly useful for use in cell therapy, drug screening, cellular research and other fields. Prior art discloses methods for in vitro differentiation of primate pluripotent stem (pPS) cells into several select cell types without prior formation of embryoid bodies.

In US 2002/019046, differentiation of pPS cells is effected directly by plating sub-confluent cultures of pPS cells onto a solid surface that facilitates differentiation, in the absence of feeder cells or in culture conditions that simulate the presence of feeder cells. However, in US 2002/019046 only cells having the phenotypic markers of hepatocytes, neurons or glia were obtained, whereas mesenchymal cells were not reported and were in fact considered undesired.

Mesenchymal progenitor or stem cells (MPC or MSC) have been previously isolated from adult tissues, e.g., bone marrow (Pittenger et al. 1999. Science 284: 143-7), adipose tissue (Zuk et al. 2001. Tissue Eng 7: 211-28) and dermis or other connective tissues (Young et al. 2001. Anat Rec 264: 51-62). MPC and/or MSC cells can be cultured and expanded in vitro, where they usually form a monolayer and display a stable phenotype, and can be induced in suitable conditions to differentiate into cells of mesenchymal lineages, such as, the osteocytic (bone), chondrocytic (cartilage), myocytic (muscle), tendonocytic (tendon), fibroblastic (connective tissue), adipocytic (fat) or stromogenic (marrow stroma) lineages (Pittenger et al. 1999; Wakitani et al. 1995. Muscle Nerve 18: 1417-26; Gang et al. 2004. Stem Cells 22: 617- 24). Multipotent MPC and/or MSC as well as more committed mesenchymal progenitors, e.g., mesenchymal progenitors committed towards differentiation along one or more than one differentiation pathways, represent a promise for cell therapy of disorders adversely affecting respective tissues of mesodermal origin, e.g., bone, cartilage, tendon, skeletal or smooth muscle, adipose tissue, or marrow stroma, or other tissues, e.g., glial or neuronal tissue. For example, multipotent MPC and/or MSC or more committed mesenchymal progenitors can be administered, e.g., injected, transplanted, applied onto, or provided in conjunction with a suitable substrate or implant, to the respective tissues of patients suffering from such diseases, whereupon the MPC and/or MSC would undergo differentiation in vivo to form cells of the respective tissues. In another example, MPC and/or MSC may be first treated in vitro to commit to or at least partly differentiate into a cell lineage needed for repairing the defect in a given patient, and the resulting cells may then be administered.

Non-limiting examples of the potential uses of MPC and/or MSC, or cells derived therefrom, in therapy include Rasulov et al., who described the use of allogeneic bone marrow derived MSC onto the surface of deep thermal burn in a human patient, leading to accelerated wound regeneration in the presence of active neo-angiogenesis (Bull Exp Biol Med 139: 141-4, 2005). In another example, Sakai et al. showed that transplantation of autologous bone marrow derived MSC led to regeneration of inter-vertebral discs in a rabbit model of disc degeneration (Biomaterials 27: 335-45, 2006). Nagaya et al. transplanted isogenic MSC isolated from bone marrow of adult rats and expanded ex vivo in a rat model of dilated cardiomyopathy (DCM). MSC transplantation increased capillary density and decreased the collagen volume fraction in the myocardium, possibly through induction of myogenesis and angiogenesis, as well as by inhibition of myocardial fibrosis (Circulation 112: 1128-35, 2005). Similarly, Amado et al. reported delivery of allogeneic bone marrow derived MSC into infracted myocardium of pigs using percutaneous-injection catheter. The transplantation of MSC resulted in long-term engraftment, reduction in scar formation and near-normalization of cardiac function (Proc Natl Acad Sci USA 102: 11474-9, 2005). Alhadlaq et al. encapsulated adipogenic cells derived from adult human MSC in a biocompatible hydrogel system to engineer adipose tissue constructs with predefined shape and dimensions, potentially utilizable in soft tissue augmentation and reconstruction, e.g., in plastic and reconstructive surgery (Tissue Eng 11 : 556-66, 2005). In another study, Guo et al. seeded culture-expanded autologous MSC into bio-ceramic scaffold beta-tricalcium phosphate (beta-TCP) in a successful attempt to repair articular cartilage defects in sheep (Tissue Eng 10: 1818-29, 2004). Further, Kawaguchi et al. isolated and expanded in vitro autologous bone marrow derived MSC from beagle dogs and used such in conjunction with atelocollagen for transplantation into experimental Class III periodontal osseous defects. The so treated defects were regenerated with cementum, periodontal ligament, and alveolar bone (J Periodontol 75: 1281-7, 2004). Quarto et al. demonstrated repair of large bone defects in humans by implantation of macroporous hydroxyapatite scaffold implants containing deposited thereon autologous osteo-progenitors derived from bone marrow MSC expanded in vitro (N Engl J Med 344: 385-6, 2001 ). Similar findings were reported in animal models (e.g., see Kon et al. 2000. J Biomed Mater Res 49: 328-37). The above non-limiting examples illustrate the considerable potential of MSC and cell types derived therefrom in tissue engineering, therapy and surgery. Other uses of MSC can be envisaged, e.g., in research of mechanisms underlying differentiation pathways of MSC into mesodermal or other cell lineages and cell types, or in research regarding the biochemical, structural or cell biological properties of differentiated cell types prepared from MSC, wherein such cell types may carry one or more mutations (e.g., heritable, targeted, induced, etc.), may be transfected with genes of interest, or the like.

However, harvesting MPC and/or MSC or more committed mesenchymal progenitors from adult tissues, e.g., bone marrow, adipose tissue or other connective tissues, requires invasive procedures in a patient or (provided such is available) in a suitable donor. Moreover, the number of MPC and/or MSC or more committed mesenchymal progenitors that can be obtained from a single donor is limited. Therefore, extensive proliferation of MPC and/or MSC or more committed mesenchymal progenitors in vitro may be requisite in order to arrive at sufficient numbers of such cells for differentiation and/or transplantation. However, the capacity of adult derived MSC or committed progenitors for long-term proliferation may not be optimal. For example, Banfi et al. (Exp Hematol 28: 707-15, 2000) reported that in vitro culturing of bone marrow MSC decreases their proliferation potential as well as their capability to undergo differentiation when treated with growth factors like FGF-2. In that study, bone forming efficiency of in vitro cultured bone marrow MSC was decreased 36 times already at first passage, compared to freshly isolated bone marrow. With view on resolving the above problems, a method which could derive substantial numbers of MPC and/or MSC or more committed mesenchymal progenitors from pluripotent stem (PS) cells, such as ES cells, would advantageously provide for a high capacity source of MPC and/or MSC and specialised cells derived therefrom. Barberi et al. (PLoS Med 2: e161 , 2005) describes a method for generation and purification of multipotent mesenchymal precursors from human ES cells. Therein, undifferentiated hES cells are plated on a monolayer of murine OP9 stromal cells. After 40 days, a heterogeneous cell population is obtained, comprising about 5% of CD73+ MSC, which are subsequently isolated using fluorescence sorting and further expanded in vitro. Barberi et al. 2005 does not disclose isolation of mesenchymal progenitors committed towards differentiation along the osteogenic pathway.

WO 2003/04605 describes a method of differentiation of hES cells to cell populations comprising mesenchymal cells of various types, and in particular the generation of a telomerised osteogenic cell line (HEF1 ). However, in the method of 2003/04605 the differentiation process is initiated through embryoid bodies, which entails disadvantages as described above.

In view of the above discussion, there remains a great need in the art for a relatively simple and reproducible method for generation of cell populations comprising considerable proportion of MPC and/or MSC, and particularly mesenchymal progenitors capable of and/or committed towards differentiation along the osteogenic pathway, starting from primate, esp. human, pluripotent stem cells.

Summary of the Invention The present invention provides a method for differentiating primate, in particular human, pluripotent stem cells into mesenchymal progenitor and/or stem cells (MPC and/or MSC) or like cells, and preferably mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway.

In an aspect, a method for differentiating primate pluripotent stem (pPS) cells into mesenchymal progenitor and/or stem cells (MPC and/or MSC), preferably mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway according to the invention comprises the steps: a) plating undifferentiated pPS cells onto a substrate which allows adherence of cells thereto, b) culturing the pPS cells of a) which have adhered to the said substrate in a medium comprising serum or plasma and allowing differentiation of the pPS cells, c) passaging the cells obtained in b) at least one time and preferably two or more times, wherein the cells are replated onto a substrate which allows adherence of cells thereto and cultured in a medium comprising serum or plasma and allowing for differentiation of the said cells. The method can further comprise a step of harvesting the so obtained cell population comprising MPC and/or MSC or like cells, preferably mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway, and optionally enriching the said cell population for or isolating said cells there from.

Importantly, the method of the present invention does not necessitate any directed enrichment for cells showing particular characteristics other than the ability to adhere to a substrate and grow under the specified conditions. Hence, the desired selection of mesenchymal progenitor cells is presently achieved through the advantageous combination of culturing in adherent conditions and re-plating. This sets the current method well apart from previously known methods which rely on selecting a desired cell type using a suitable cell- sorting method, such as FACS cell sorting, cell panning or affinity isolation, based on the cell- type's properties, usually the expression of specific cell surface markers thereby. In contrast, the present method does not require such directed isolation of a specific subset of cells to eventually arrive at a substantially homogeneous population of mesenchymal progenitors, thereby providing for considerable simplification. By means of example, EP 1 627 913 discloses a method wherein obtaining mesenchymal stem cells from pluripotent stem cells requires to isolate a specific small subset of PDFGRα-positive and FLK-1 negative cells. The present method clearly does not need and thus does not comprise such a selection step.

Hence, in the method of the invention, cells re-plated in a particular passaging step are representative of substantially all adherent cells subjected to said passaging, i.e., are representative of substantially all adherent cells resulting from the previous passage (i.e., apart from any enrichment that might be inherent to the methods of detaching, disassociating and re-plating the cells during a passaging step, no further intentional selection of a particular cell subset(s) is necessitated in the method; hence, the composition of the re-plated cell population is similar or identical to the composition of the cell population subjected to passaging). For example, in step b) cells representative of substantially all cells that have adhered to the substrate after plating of the undifferentiated pPS cells in step a) are subjected to culturing; similarly, in step c) cells representative of substantially all cells which have adhered to the substrate and were sustained in step b) are passaged and re-plated; similarly, on each subsequent passage of step c) cells representative of substantially all cells which have adhered to the substrate and were sustained after the previous passage are passaged and re-plated.

As shown by the experimental evidence, the method provides a simple and reliable way to obtain cell populations having a considerable proportion of cells having MPC and/or MSC characteristics, and preferably of mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway, or even substantially homogeneous populations of MPC and/or MSC cells, and preferably of mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway, while not requiring the use of embryoid bodies to initiate differentiation, avoiding co-culture with other cells, and especially cells of other origin than the pPS cells, and not requiring per se the addition of exogenous growth factors, i.e., growth factors other than those present in the serum. The obtained MPC and/or MSC, and preferably mesenchymal progenitor cells committed towards differentiation along the osteogenic pathway, and cell populations, show a characteristic fibroblast-like, mesenchymal cell morphology, display a multitude of mesenchymal-specific marker molecules and demonstrate ability of differentiation into cells of at least one and potentially more mesodermal cell lineages, preferably into at least or only the osteogenic lineage. Potentially, the cells may display plasticity or transdifferentiation towards at least neuronal-like cells, as judged, e.g., by morphological criteria and/or marker expression. In a particularly preferred embodiment, the present method provides for mesenchymal progenitor cells and populations that can be differentiated into cells of at least the osteogenic lineage.

In a further embodiment, the method provides for mesenchymal progenitor cells and populations that can be differentiated into cells of (at least) the osteogenic lineage, but not to cells of the chondrogenic and/or adipogenic lineages. This limited differentiation potential of such mesenchymal progenitor cells of this embodiment may be useful, e.g., in applications where the formation of other than bone cell types would be undesirable, such as, for example in bone tissue engineering or bone reconstitution by cell transplantation, etc.).

Accordingly, in an embodiment, the method provides for mesenchymal progenitor cells committed towards differentiation along the osteogenic pathway ("osteoprogenitor cells" or

"bone progenitor cells"). In an example, such cells may express one or more markers of osteoblastic differentiation, e.g., one or more markers of an early stage of osteoblastic differentiation, such as, for example, CBFA1/RUNX2 and/or collagen type I. Such cells may also express markers attributable to more differentiated osteoblasts or osteocytes, such as bone-type alkaline phosphatase (ALP) and osteocalcin (OC).

By providing considerable numbers of MPC and/or MSC, and particularly preferably of mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway, which are directly derived from pPS cells, e.g., human ES cells, the present method will further enable therapeutic applications, such as tissue engineering and cell therapy applications. In addition, pPS cells may be advantageously used for the present differentiation, such as to yield MPC and/or MSC, and particularly preferably mesenchymal progenitor cells capable of and/or committed towards differentiation to osteogenic lineage, which would be well tolerated by the patient's immune system.

Further aspects of the invention relate to isolated MPC and/or MSC, and preferably mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway, and cell populations obtainable or directly obtained using the methods, to cell types, esp. mesodermal tissue cell types, particularly preferably osteogenic cell types, obtainable or directly obtained by differentiating the isolated MPC and/or MSC, or preferably osteoprogenitor cells, or cell populations of the invention, and to uses of such MPC and/or MSC, and preferably of osteoprogenitors, or further differentiated cells in therapy, research, drug screening, and other applications. These and other features of the invention are further explained here below and stated in the claims, as well as illustrated by non-limiting examples.

Brief Description of the Figures

Figure 1 illustrates growth of undifferentiated human ES cells in colonies (H) and splitting of such colonies for further culture using mechanical dissociation as described by Mateizel et al. 2006 (G).

Figure 2 illustrates cell morphology after 24 days following the plating of pPS (hES) cells for differentiation into mesenchymal progenitors according to the methods of the invention. A, B, D show different optical fields at 10Ox magnification. C shows an enlargement of the optical field of B at 20Ox magnification. Figure 3 illustrates cell morphology after the cells of figure 2 were subjected to further passages according to the invention. Magnification 10Ox. The bottom photograph displays cells having higher confluence.

Figure 4 shows FACS assessment of surface markers in mesenchymal progenitors obtained from hES cells according to the invention.

Figure 5 illustrates quantification of vimentin (white columns) and a-SMA (black columns) in mesenchymal progenitor cells obtained according to the invention, relative to a primary culture of bone-marrow derived human mesenchymal stem cells (Pittenger et al. 1999). Y- axis: relative quantification (log 10). Figure 6 illustrates osteogenic differentiation of mesenchymal progenitors obtained from hES cells according to the invention. Phase contrast images of calcium accumulation in samples treated with induction medium (β-glycerol phosphate, dexamethasone and ascorbic acid) (Figure 6A-C, 4Ox) compared with the controls (Figure 6D-F, 4Ox). A, D: hMSCVUBOI ; B, E: hMSCVUB02; C,F: hMSCVUB03_DM1. Figure 7 (A) illustrates representative cytoplasmic vimentin staining (immuno-cytochemistry) for mesenchymal progenitor cells obtained from hES cells according to the invention (scale bar: 100 μm); (B) illustrates relative quantification by real-time RT-PCR of POU5F-1 and NANOG mRNA expression in mesenchymal progenitor cells obtained from hES cells according to the invention. For each gene, expression was made relative to the corresponding undifferentiated hES cells (value of zero) and was corrected for GAPDH. Error bars represent standard deviation for three PCR replicates.

Figure 8 illustrates characterisation of osteoblast or osteogenic markers in representative mesenchymal progenitor cells obtained from hES cells according to an embodiment of the invention: (A, B) relative quantification by real-time RT-PCR of (A) CBFA1/RUNX2 and (B) osteocalcin (OCN) mRNA expression. For each gene, expression was made relative to the corresponding undifferentiated hES cells (value of one) and was corrected for GAPDH. Error bars represent standard deviation for three PCR replicates. (C, D) Immuno-cytochemistry of the above MPC and/or MSC cells showing nuclear expression of CBFA1/RUNX2 (C) and cytoplasmic localization of collagen type 1 (D) (scale bar: 100 μm). VUB01 -MSCL denotes hMSCVUBOI , VUB02-MSCL denotes hMSCVUB02, and VUB03_DM1-MSCL denotes hMSCVUB03-DM1 ; VUB01 , VUB02 and VUB03_DM1 denote the respective undifferentiated hES cell lines. Figure 9 illustrates characterisation of osteoblastic and osteocytic markers (CBFA1 , COLLI , ALP and OCN) in representative mesenchymal progenitor cells obtained from hES cells according to an embodiment of the invention and further differentiated in osteogenic medium, by quantitative RT-PCR. Figure 10 illustrates characterisation of ALP in representative mesenchymal progenitor cells obtained from hES cells according to an embodiment of the invention and further differentiated in osteogenic medium, using immunohistochemistry.

Figure 11 illustrates transdifferentiation, as judged by morphological criteria, of the mesenchymal progenitor cells obtained from hES cells according to the invention into neuronal cells. (A,B) Phase contrast images (10Ox) of samples treated with the induction medium (A) compared with the control (B).

Detailed Description of the Invention

As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a cell" refers to one or more than one cell.

The terms "comprising", "comprises" and "comprised of as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.

All references cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings of all references herein specifically referred to are incorporated by reference.

For general methods relating to the invention, reference is made to well-known textbooks, including, e.g., "Molecular Cloning: A Laboratory Manual, 2nd Ed." (Sambrook et al., 1989), Animal Cell Culture (R. I. Freshney, ed., 1987), the series Methods in Enzymology (Academic Press), Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds., 1987); "Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Ed." (F. M. Ausubel et al., eds., 1987 & 1995); Recombinant DNA Methodology Il (R. Wu ed., Academic Press 1995), incorporated by reference herein.

For further elaboration of general techniques useful in the practice of this invention, the practitioner can refer to standard textbooks and reviews in cell biology, tissue culture, and embryology. Included are "Teratocarcinomas and embryonic stem cells: A practical approach" (E. J. Robertson, ed., IRL Press Ltd. 1987); "Guide to Techniques in Mouse Development" (P. M. Wasserman et al. eds., Academic Press 1993); "Embryonic Stem Cell Differentiation in Vitro" (M. V. Wiles, Meth. Enzymol. 225:900, 1993); "Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy" (P. D. Rathjen et al., al.,1993). Differentiation of stem cells is reviewed, e.g., in Robertson. 1997. Meth Cell Biol 75: 173; and Pedersen. 1998. Reprod Fertil Dev 10: 31 , incorporated by reference herein.

General techniques in cell culture and media collection are outlined in Large Scale Mammalian Cell Culture (Hu et al. 1997. Curr Opin Biotechnol 8: 148); Serum-free Media (K. Kitano. 1991. Biotechnology 17: 73); Large Scale Mammalian Cell Culture (Curr Opin Biotechnol 2: 375, 1991), incorporated by reference herein.

In an aspect, the invention relates to a method for in vitro differentiating primate pluripotent stem (pPS) cells into mesenchymal progenitor and/or stem cells (MPC and/or MSC), particularly preferably into mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway, comprising the steps: a) plating undifferentiated pPS cells onto a substrate which allows adherence of cells thereto, b) culturing the pPS cells of a) which have adhered to the said substrate in a medium comprising serum or plasma and allowing differentiation of the pPS cells, c) passaging the cells obtained in b) at least one time and preferably two or more times, wherein the cells are replated onto a substrate which allows for adherence of cells thereto and cultured in a medium comprising serum or plasma and allowing for differentiation of the said cells. The method can further comprise a step of harvesting the so obtained cell population comprising MPC and/or MSC or like cells, preferably harvesting the mesenchymal progenitors capable of and/or committed towards differentiation along the osteogenic pathway, and optionally enriching the said cell population for or isolating of MPC and/or MSC or like cells, preferably said osteogenic mesenchymal progenitors, there from.

The term "progenitor cell" refers generally to an unspecialised or relatively less specialised and proliferation-competent cell, which or the progeny of which can give rise to at least one relatively more specialised cell type. By means of example and not limitation, a progenitor cell may give rise to descendants that can differentiate along one or more lineages to produce increasingly relatively more specialised cells, wherein such descendants and/or increasingly relatively more specialised cells may themselves be progenitor cells as defined herein, or even to produce terminally differentiated cells, i.e., fully specialised cells, which may be postmitotic. The term also encompasses stem cells as defined herein.

The term "stem cell" refers to a progenitor cell as defined herein, which is further capable of self-renewal, i.e., can proliferate without differentiation. The term encompasses stem cells capable of substantially unlimited self-renewal, i.e., wherein the progeny of a stem cell or at least part thereof substantially retains the unspecialised or relatively less specialised phenotype, the differentiation potential, and the proliferation capacity of the mother stem cell, as well as stem cells which display limited self-renewal, i.e., wherein the capacity of the progeny or part thereof for further proliferation and/or differentiation is demonstrably reduced compared to the mother cell.

A skilled person knows that the above properties generally refer to the in vivo behaviour of progenitor cells and stem cells, and may under appropriate conditions be completely or at least in part replicated in vitro and/or ex vivo. By means of illustration and not limitation, isolation and substantially unlimited in vitro cultivation of pluripotent human embryonic stem (ES) cells without differentiation has been described by Thomson et al. (Science 282: 1145- 1147, 1998) and US 6,200,806..

A progenitor or stem cell is said to "give rise" to another, more differentiated or relatively more specialised, cell when, for example, the progenitor or stem cell differentiates to become the other cell without previously undergoing cell division, or if the other cell is produced after one or more rounds of cell division and/or differentiation of the progenitor or stem cell or progeny thereof.

Based on the ability to give rise to diverse cell types, progenitor and/or stem cells are usually described as totipotent, pluripotent, or multipotent. A single "totipotent" progenitor and/or stem cell is defined as being capable of growing, i.e. developing, into an entire organism. A "pluripotent" progenitor and/or stem cell is not able of growing into an entire organism, but is capable of giving rise to cell types originating from all three germ layers, i.e. mesoderm, endoderm, and ectoderm, and may be capable of giving rise to all cell types of an organism. A "multipotent" progenitor and/or stem cell is capable of giving rise to at least one cell type from each of two or more different organs or tissues of an organism, wherein the said cell types may originate from the same or from different germ layers, but is not capable of giving rise to all of the cell types of an organism. A "unipotent" progenitor and/or stem cell is capable of differentiating to cells of only one cell lineage. Prototype "primate pluripotent stem cell" or "pPS cell" is a pluripotent cell derived from any kind of primate embryonic tissue, e.g., foetal or pre-foetal tissue, the cell being capable under appropriate conditions of producing progeny of different cell types that are derivatives of all three germinal layers, i.e., endoderm, mesoderm, and ectoderm, according to a standard art- accepted test, such as the ability to form a teratoma after 8 weeks in SCID mice, or the ability to form identifiable cells of all three germ layers in tissue culture.

Included in the definition of pPS cells are embryonic cells of various types, exemplified by human embryonic stem (hES) cells, e.g., as described by Thomson et al. (Science 282:1145- 1147, 1998), human embryonic germ (hEG) cells, e.g., as described by Shamblott et al. (Proc Natl Acad Sci USA 95: 13726, 1998), embryonic stem cells from other primates, such as, Rhesus stem cells, e.g., as described by Thomson et al. (Proc Natl Acad Sci USA 92:7844- 7848, 1995) or marmoset stem cells, e.g., as described by Thomson et al. (Biol Reprod 55: 254-259, 1996). Other types of primate pluripotent cells are also included in the term as are any cells of primate origin that are capable of producing progeny that are derivatives of all three germinal layers, regardless of whether they were derived from embryonic tissue, foetal tissue, or other sources. pPS cells are not derived from a malignant source. A cell or cell line is from a "non-malignant source" if it was established from primary tissue that is not cancerous, nor from a cell that was genetically altered with a known oncogene. Typically, immortalisation of cells by telomerisation maintains their non-malignant status. It is desirable, but not always necessary, that the pPS cells have a normal karyotype.

As noted, prototype "human ES cells" ("hES cells") are described by Thomson et al. 1998 and in US 6,200,806. The scope of the term covers pluripotent stem cells that are derived from a human embryo at the blastocyst stage, or before substantial differentiation of the cells into the three germ layers. The hES cells are typically derived from the inner cell mass of blastocysts or from whole blastocysts. Derivation of hES cell lines from the morula stage has been documented and ES cells so obtained can also be used in the invention (Strelchenko et al. 2004. Reproductive BioMedicine Online 9: 623-629).

As noted, prototype "human EG cells" ("hEG cells") are described by Shamblott et al. 1998. Such cells may be derived, e.g., from gonadal ridges and mesenteries containing primordial germ cells from foetuses. In humans, the foetuses may be typically 5-11 weeks post- fertilization. Those skilled in the art will appreciate that, except where explicitly required otherwise, the terms pPS cells, hES cells and hEG cells include primary tissue cells and established lines that bear phenotypic characteristics of the respective cells, and derivatives of such primary cells or cell lines that still have the capacity of producing progeny of each of the three germ layers.

Exemplary established lines of human ES cells include lines which are listed in the NIH Human Embryonic Stem Cell Registry (http://stemcells.nih.gov/research/registry), and sublines thereof, such as, lines hESBGN-01 , hESBGN-02, hESBGN-03 and hESBGN-04 from Bresagen Inc. (Athens, GA), lines Sahlgrenska 1 and Sahlgrenska 2 from Cellartis AB (Goteborg, Sweden), lines HES-1 , HES-2, HES-3, HES-4, HES-5 and HES-6 from ES Cell International (Singapore), line Miz-hES1 from MizMedi Hospital (Seoul, Korea), lines I 3, I 3.2, I 3.3, I 4, I 6, I 6.2, J 3 and J 3.2 from Technion - Israel Institute of Technology (Haifa, Israel), lines HSF-1 and HSF-6 from University of California (San Francisco, CA), lines H1 , H7, H9, H13, H14 of Wisconsin Alumni Research Foundation / WiCeII Research Institute (Madison, Wl), lines CHA-hES-1 and CHA-hES-2 from Cell & Gene Therapy Research Institute / Pochon CHA University College of Medicine (Seoul, Korea), lines H1 , H7, H9, H13, H14, H9.1 and H9.2 from Geron Corporation (Menlo Park, CA), lines Sahlgrenska 4 to Sahlgrenska 19 from Goteborg University (Goteborg, Sweden), lines MB01 , MB02, MB03 from Maria Biotech Co. Ltd. (Seoul, Korea), lines FCNCBS1 , FCNCBS2 and FCNCBS3 from National Centre for Biological Sciences (Bangalore, India), and lines RLS ES 05, RLS ES 07, RLS ES 10, RLS ES 13, RLS ES 15, RLS ES 20 and RLS ES 21 of Reliance Life Sciences (Mumbai, India). Other exemplary established hES cell lines include those deposited at the UK Stem Cell Bank (http://www.ukstemcellbank.org.uk/), and sub-lines thereof, e.g., line WT3 from King's College London (London, UK) and line hES-NCL1 from University of Newcastle (Newcastle, UK) (Strojkovic et al. 2004. Stem Cells 22: 790-7). Further exemplary ES cell lines include lines FC018, AS034, AS034.1 , AS038, SA111 , SA121 , SA142, SA167, SA181 , SA191 , SA196, SA203 and SA204, and sub-lines thereof, from Cellartis AB (Goteborg, Sweden).

Establishment of other exemplary hES cell lines, which (or sub-lines thereof) may be used in the context of the present invention, has been published, e.g., in Mateizel et al. (Hum Reprod 21 : 503-11 , 2006) - 5 lines (2 genetically normal: VUB01 , VUB02; 3 carrying a genetic mutation: VUB03_DM1 , VUB04_CF and VUB05JHD), Findikli et al. (Reprod Biomed Online 10: 617-27, 2005) - 7 lines, Hong Chen et al. (Human Reproduction 20: 2201 -2206, 2005) - 2 lines, Genbacev et al. (Fertil Steril 83: 1517-29, 2005), Pickering et al. (Reproductive BioMedicine Online 10: 390-397, 2005) - 1 line with cystic fibrosis mutation, Oh et al. (Stem Cells 23: 211-9, 2005) - 3 lines: SNUhESI , SNUhES2 and SNUhES3, Simon et al. (Fertil Steril 83: 246-9, 2005) - 2 lines, Lee et al. (Biol Reprod 72: 42-9, 2005) - 3 lines: Miz-hES-14, Miz-hES-15 and Miz-hES-9, Verlinsky et al. (Reproductive BioMedicine Online 10: 105-110, 2005) - 18 lines carrying mutations responsible for genetic disorders, Strelchenko et al. 2004 - 8 lines derived from morula stage, Baharvand et al. (Differentiation 72: 224-9, 2004) - 1 line, Cowan et al. (N Engl J Med 350: 1353-1356, 2004) - 17 lines: HUES1 through HUES17, Park et al. (Hum Reprod 19: 676-84, 2004) - 9 lines, Park et al. (Biol Reprod 69: 2007-14, 2003) - 3 lines: Miz-hES1 , Miz-hES2, and Miz-hES3, Pickering et al. (Reprod Biomed Online 7: 353-64, 2003) - 1 line, Hovatta et al. (Hum Reprod 18: 1404-9, 2003) - 2 lines and Richards et al. (Nat Biotechnol 20: 933-6, 2002) - 1 line.

A skilled person will appreciate that further cell lines having characteristics of primate, esp. human, pluripotent cells, esp. ES cells (e.g., hES cells) or EG cells (e.g., hEG cells), may be established in the future, and these may too be suitable in the present invention. A skilled person can also use techniques known in the art to verify that the above hES cell lines, or other existing or yet to be established pPS cell lines, or sub-lines thereof, show desirable cell characteristics, such as expansion in vitro in undifferentiated state, preferably normal karyotype and ability of pluripotent differentiation. Within the present specification, the term "differentiation", "differentiating" or derivatives thereof denote the process by which an unspecialised or relatively less specialised cell, such as, for example, a pluripotent stem cell or the progeny or potential progeny of such a stem cell, becomes relatively more specialised. In the context of cell ontogeny, the adjective "differentiated" is a relative term. Hence, a "differentiated cell" is a cell that has progressed further down a certain developmental pathway than the cell it is being compared with. The differentiated cell may, for example, be a "terminally differentiated cell", i.e., a fully specialised cell which takes up specialised functions in various tissues or organs of an organism, which may but need not be post-mitotic; or the differentiated cell may also be a progenitor cell within a particular differentiation lineage, which can further proliferate and/or differentiate. Similarly, a cell is "relatively more specialised" if it has progressed further down a certain developmental pathway than the cell it is being compared with, wherein the latter is therefore considered "unspecialised" or "relatively less specialised". A relatively more specialised cell may differ from the unspecialised or relatively less specialised cell in one or more demonstrable phenotypic characteristics, such as, for example, the presence, absence or level of expression of particular cellular components or products, e.g., RNA, proteins or other substances, activity of certain biochemical pathways, morphological appearance, proliferation capacity and/or kinetics, differentiation potential and/or response to differentiation signals, etc., wherein such characteristics signify the progression of the relatively more specialised cell further along the said developmental pathway. Non-limiting examples of differentiation may include, e.g., the change of a pluripotent stem cell into a given type of multipotent stem cell, the change of a multipotent stem cell into a given type of unipotent stem cell or progenitor cell, or the change of a unipotent stem cell or progenitor cell to more specialised cell types or to terminally specialised cells within a given cell lineage. Differentiation of an unspecialised or less specialised cell to a more specialised cell may proceed through appearance of cells with an intermediate degree of specialisation.

The terms "mesenchymal progenitor cell" or "MPC" as used herein refer interchangeably to a multipotent or unipotent, progenitor cell capable of generating cells of mesenchymal cell lineages, usually cells of at least one, two or more mesenchymal cell lineages, e.g., osteocytic (bone), chondrocytic (cartilage), myocytic (muscle), tendonocytic (tendon), fibroblastic (connective tissue), adipocytic (fat) or stromogenic (marrow stroma) cell lineage. For example, a cell may be considered MPC if it is capable of forming cells of at least one, two or three lineages chosen from the adipocytic, chondrocytic, and osteocytic lineage, using standard, art-accepted differentiation conditions and cellular phenotype evaluation methods, e.g., as described in Pittenger et al. 1999 or Barberi et al. 2005. By means of a preferred example and not limitation, a cell may be considered MPC herein if it is capable of forming cells of at least the osteogenic lineage. The term encompasses the progeny of MPC, e.g., progeny obtained by in vitro or ex vivo propagation of MPC, esp. without differentiation. The terms also encompass "mesenchymal stem cells" or "MSC", which refer to mesenchymal progenitor cells capable of at least limited self-renewal, such as, e.g., self-renewal in vivo, in vitro or ex vivo, and progeny thereof.

Cells referred to as MPC and/or MSC have been typically isolated from adult tissues in the art. The present invention provides a method for differentiating pPS cells into cells having at least select characteristics of mesenchymal progenitor or stem cells. In particular, such characteristics can relate to the potential of these cells for differentiation into one or more mesenchymal lineages, as described above. Another preferred feature of the obtained cells may be their at least limited self-renewal capacity, as described above. Further, MPC and/or MSC, and particularly preferably mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway, obtainable according to the invention from pPS cells may but need not display also other characteristics typical of MPC and/or MSC isolated from adult tissues, such as, e.g., morphology, growth characteristics and/or marker profile, as discussed below. Given the possible phenotypic differences between the pPS-derived cells of the invention and the adult-derived progenitors referred to as MPC and/or MSC in the art, the cells of the invention may optionally be referred to as MPC-like and/or MSC-like cells. However, in the present specification, the said cells are generally referred to as MPC and/or MSC, in particular on basis of their differentiation potential and/or at least limited capacity to proliferate without differentiation, as defined above.

The methods of the present invention produce MPC and/or MSC, preferably mesenchymal progenitor cells capable of and/or committed toward differentiation along the osteogenic pathway, by differentiating primate pluripotent cells or cell lines, in particular, hES or hEG cells or cell lines, or sub-lines thereof. Accordingly, in the present specification, unless stated otherwise, the origin (i.e., correspondence to a particular species of organisms) of the MPC and/or MSC is determined by the origin of the pPS cells or cell lines from which the MPC and/or MSC are derived.

MPC and/or MSC in general, and as produced by the methods of the present invention in particular, may be characterized by a number of other phenotypic criteria. For example, methods are known to maintain and expand, at least to a certain extent, undifferentiated MPC and/or MSC in vitro (see, e.g., Banfi et al. 2000). Such MPC and/or MSC retain the ability of (multipotent) differentiation into one or more mesenchymal cell lineages (esp. as above) and, preferably but not necessarily, a normal karyotype. Morphologically, such undifferentiated mesenchymal cells are usually recognized by their growth in monolayers and their characteristic mononuclear ovoid, stellate shape or spindle shape, with a round to oval nucleus. The oval elongate nuclei typically have prominent nucleoli and a mix of heterochromatin and euchromatin. The cells have little cytoplasm but many thin processes that appear to extend from the nucleus. It is further believed that MPC and/or MSC, e.g., human MSC, may typically stain for one, two, three or more of the following markers: CD73 (SH3, SH4), CD106 (VCAM), CD166 (ALCAM), CD90, CD105 (SH2), CD29, CD44, CD54, GATA-4, and alkaline phosphatase, while being negative for hematopoietic lineage cell markers (e.g., CD14, CD34 and CD45). MPC and/or MSC may also express STRO-1 as a marker.

MPC and/or MSC, esp. human MSC, with at least some of the above characteristics have been previously isolated from adult tissues, e.g., from bone marrow, blood, umbilical cord, placenta, foetal yolk sac, skin (dermis), specifically foetal and adolescent skin, periosteum or adipose tissue. For example, isolation of MPC and/or MSC from adult tissues, in vitro expansion thereof without differentiation, phenotypic characterisation of the MPC and/or MSC, and differentiation of MPC and/or MSC have been described, e.g., in Pittenger et al. 1999, Banfi et al. 2000, US 5,486,359; US 5,811 ,094; US 5,736,396; US 5,837,539 or US 5,827,740. MPC and/or MSC obtainable by the methods of the invention may also have phenotypic characteristics identical to or at least in part similar to such MPC and/or MSC isolated from adult tissues.

The term "cell population" generally refers to a grouping of cells. A cell population may consist of or may comprise at least a fraction of cells of a common type, or having characteristics in common. Such characteristics may include, without limitation, morphological characteristics, potential for differentiation (e.g., pluripotent, multipotent, unipotent, etc.; e.g., if multipotent or unipotent, ability to differentiate towards specific cell types), or the presence and/or level of one, two, three or more cell-associated markers, e.g., surface antigens. Such characteristics may thus define a cell population or a fraction thereof. The term "cell population comprising MPC and/or MSC or mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway" refers to a cell population as defined herein comprising respectively at least a fraction of MPC and/or MSC or of mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway as defined herein. Cells in the said fraction may, but need not be, of an identical phenotype. By means of illustration, but not limitation, the said fraction of MPC and/or MSC may comprise multipotent MPC and/or MSC capable of forming cells of at least one and preferably at least two mesenchymal cell lineages, as well as proliferative precursor cells committed to form cells of a particular mesenchymal tissue or lineage. In another non-limiting example, the said fraction may comprise cells which are identical in or which differ to various extent in their staining pattern with regard to cell markers considered to be specific. Preferably, when a cell population comprising MPC and/or MSC is exposed to specific differentiation conditions known per se in the art, the said fraction of MPC and/or MSC is capable of giving rise to cells of at least one, or at least two mesenchymal cell lineages, or even three or more mesenchymal cell lineages, e.g., of osteocytic (bone), chondrocytic (cartilage), myocytic (muscle), tendonocytic (tendon), fibroblastic (connective tissue), adipocytic (fat) or stromogenic (marrow stroma) cell lineage. It is to be understood that this overall capability of the MPC and/or MSC fraction does not imply that each cell constituting the fraction would need to possess, on its own, the ability to differentiate to cells of at least one particular, or at least two, preferably three or more mesenchymal cell lineages. Rather, this overall capability of the MPC and/or MSC fraction may reflect the sum of the potentially differing individual differentiation capabilities of the cells constituting the said fraction. Nevertheless, a situation in which all or a proportion, e.g., a considerable proportion, of the cells of the MPC and/or MSC fraction do possess such ability, is also contemplated.

The term "substantially homogeneous population of MPC and/or MSC, or preferably of mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway" denotes a cell population comprising respectively MPC and/or MSC or said bone-committed mesenchymal progenitor cells as defined above, wherein said fraction in the said cell population is at least 50%, e.g., at least 55%, preferably at least 60%, e.g., at least 65%, more preferably at least 70%, e.g., at least 75%, even more preferably at least 80%, e.g., at least 85%, most preferably at least 90%, e.g., at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even close to or equal to 100%. pPS cells or cell lines and cultures thereof are described as "undifferentiated" or "substantially undifferentiated" when a substantial proportion of stem cells and their derivatives in a cell population display characteristics (e.g., morphological or markers) of undifferentiated pPS cells, clearly distinguishing them from differentiated cells of embryo or adult origin. Undifferentiated pPS cells are easily recognized by those skilled in the art, and typically appear in the two dimensions of a microscopic view with high nuclear/cytoplasmic ratios and prominent nucleoli. It is understood that colonies of undifferentiated cells within the population may often be surrounded by neighbouring cells that are more differentiated. Nevertheless, the undifferentiated colonies persist when the population is cultured or passaged under appropriate conditions, and individual undifferentiated cells constitute a substantial proportion of the cell population. Primate PS cells express the stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies designated Tra-1-60 and Tra-1-81 (Thomson et al. 1998). Undifferentiated hES cells may also typically express Oct-4 and TERT, e.g., as detected by RT-PCR. Differentiation of hES cells in vitro typically results in the loss of these markers.

Cultures comprising pPS cells, that are termed "undifferentiated" or "substantially undifferentiated" herein may contain at least 60%, preferably at least 70%, even more preferably at least 80%, e.g., at least 90% or more than 90% and up to 100% undifferentiated pPS cells (in terms percentage of cells with the same genotype that are undifferentiated).

The term "in vitro" as used herein denotes outside, or external to, animal or human body. The term "in vitro" as used herein should be understood to include "ex vivo". The term "ex vivo" typically refers to tissues or cells removed from an animal or human body and maintained or propagated outside the body, e.g., in a culture vessel.

The methods of the present invention use undifferentiated pPS cells, such as primary tissue pPS cells, pPS cell lines, sub-lines or derivatives established therefrom, or established pPS cell lines, in particular ones presently available in the art, although the use of pPS cell lines which will be established in the future is also envisaged (the above herein collectively encompassed in the term "pPS cells", as defined elsewhere).

Methods for isolating pluripotent stem cells and establishing and expanding cultures of undifferentiated pPS cells, are well known in the art. In non-limiting examples, embryonic stem cells can be isolated from blastocysts of members of the primate species, as illustrated, e.g., by Thomson et al. 1995. Human embryonic stem cells can be prepared from human blastocyst cells using the techniques described by, e.g., Thomson et al. 1998, US 5,843,780, Thomson et al. 1998"b" (Curr Top Dev Biol 38: 133) or Reubinoff et al. 2000 (Nature Biotech. 18: 399), or from morula stage as described by Strelchenko et al. 2004. Human embryonic germ cells can be prepared from foetal gonadal tissue, as described, e.g., in US 6,090,622 and by Shamblott et al. 1998. An illustrative and non-limiting example of derivation of hES cells includes, in brief, steps as follows. Human blastocysts are obtained from human in vitro fertilized embryos or alternatively from in vivo pre-implantation embryos (Bongso et al. 1989. Hum Reprod 4: 706). Embryos can be cultured to the blastocyst stage in suitable media, including but not limited to G1.2 and G2.2 medium (Gardner et al. 1998. Fertil Steril 69: 84), Cook medium, Medicult, or the like. The zona pellucida is removed from developed blastocysts by brief exposure to pronase (Sigma). The inner cell masses are isolated by immuno-surgery as known in the art, e.g., in which blastocysts are exposed to a 1 : 1 dilution of goat anti-human serum for 30 min, then briefly washed three times in hES medium, and exposed to a 1 : 5 dilution of Guinea pig complement (Gibco) for at least 10 min. After two further washes in hES medium, lysed trophectoderm cells are removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM plated on mEF feeder layers. After 9 to 15 days, inner cell mass-derived outgrowths are dissociated into clumps, either by exposure to calcium and magnesium-free phosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispase or trypsin, or by mechanical dissociation with a micropipette; and then re-plated on mEF in fresh medium. Growing colonies having undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and re-plated. ES-like morphology is characterized as compact colonies with apparently high nucleus to cytoplasm ratio and prominent nucleoli. Resulting ES cells are then routinely split every 4-7 days, e.g., by brief trypsinisation, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase (-200 U/mL; Gibco), by selection of individual colonies by micropipette, or by mechanical dissociation (Mateizel et al. 2005). Clump sizes of about 50 to 100 cells are optimal.

An illustrative and non-limiting example of derivation of hEG cells includes preparation from primordial germ cells present in human foetal material taken about 8-11 weeks after the last menstrual period (Shamblott et al. 1998, US 6,090,622). Briefly, genital ridges are rinsed with isotonic buffer, then placed into 0.1 ml_ 0.05% trypsin/0.53 mM sodium EDTA solution (BRL) and cut into <1 mm³ chunks. The tissue is then pipetted through a 100/μL tip to further disaggregate the cells. It is incubated at 37⁰C for ~5 min, then ~3.5ml_ EG growth medium is added. EG growth medium is DMEM, 4500 mg/LD-glucose, 2200 mg/L mMNaHCO3 ; 15% ES qualified foetal calf serum (BRL); 2 mM glutamine (BRL); 1 mM sodium pyruvate (BRL); 1000-2000 U/mL human recombinant leukaemia inhibitory factor (LIF, Genzyme); 1 -2 ng/mL human recombinant b-FGF (Genzyme); and 1OuM forskolin (in 10% DMSO). In an alternative approach, EG cells are isolated using hyaluronidase/collagenase/DNAse. Gonadal anlagen or genital ridges with mesenteries are dissected from foetal material, the genital ridges are rinsed in PBS, then placed in 0.1 mL HCD digestion solution (0.01 % hyaluronidase type V, 0.002% DNAse I, 0.1% collagenase type IV, all from Sigma prepared in EG growth medium). Tissue is minced, incubated 1 h or overnight at 37⁰C, re-suspended in 1-3mL of EG growth medium, and plated onto a feeder layer. Ninety-six well tissue culture plates are prepared with a sub-confluent layer of feeder cells (e.g., STO cells, ATCC No. CRL 1503) cultured for 3 days in modified EG growth medium free of LIF, b-FGF or forskolin, inactivated with 5000 rads γ-irradiation. 0.2ml_ of primary germ cell (PGC) suspension is added to each of the wells. The first passage is done after 7-10 days in EG growth medium, transferring each well to one well of a 24-well culture dish previously prepared with irradiated STO mouse fibroblasts. The cells are cultured with daily replacement of medium until cell morphology consistent with EG cells is observed, typically after 7-30 days or 1-4 passages.

A skilled person will understand that the above examples serves only to illustrate possible particular manners of hES or hEG derivation and do not limit the present invention, which mainly relates to further manipulation of so obtained cells. A skilled person will also be aware of further techniques for hES or hEG derivation which exist in the art, and is also capable of modifying and optimising such techniques.

The methods of the present invention involve the use of undifferentiated pPS cells. Provision of such cells may typically require prior in vitro propagation or expansion thereof without differentiation. Culture conditions which promote continuous proliferation of pPS cells in culture without promoting differentiation are generally known in the art and non-limiting examples are briefly addressed here below for the purposes of illustration.

Whenever a culture or cell population is referred to as proliferating "without differentiation", what is meant is that after proliferation, the composition is substantially undifferentiated according to the above definition. Populations that proliferate through at least four passages (-20 doublings) "without differentiation" may contain substantially the same proportion of undifferentiated cells (or possibly a higher proportion of undifferentiated cells) when evaluated at the same degree of confluence as the originating culture.

An exemplary, non-limiting, serum-containing hES cell medium is made with 80% DMEM (such as Knock-Out DMEM, Gibco), 20% of either defined foetal bovine serum (FBS, Hyclone) or serum replacement (WO 98/30679, i.e., serum-free conditions), 1 % non-essential amino acids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Just before use, human b- FGF is added to 4ng/ml_ (WO 99/20741 ).

Conventionally, albeit in no way limiting, ES cells are cultured on a layer of feeder cells, typically fibroblasts derived from embryonic or foetal tissue, typically mouse tissue. In a non- limiting example, embryos are harvested from a CF1 mouse at 13 days of pregnancy, transferred to 15 ml_ trypsin/EDTA, finely minced, and incubated 25 min at 37⁰C. Medium comprising 90% DMEM, 10% FBS, 1 % non essential amino acids and 2 mM glutamine was added, the debris is allowed to settle, and the cells are propagated in the same medium. To prepare a feeder cell layer, cells are treated to inhibit proliferation but permit synthesis of factors that support ES cells. Culture plates are coated with 0.1 % gelatine for 2 h, plated with 375,000 mitomycine-treated (10 μg/ml) mEF per well, and used up to 4 days after plating. The medium is replaced with fresh hES cell medium just before seeding pPS cells. Alternatively, to avoid contact and mixing between pPS cells and non-primate cells, e.g., mouse feeders, pPS cells may be cultured on primate fibroblasts. For example, Genbacev et al. 2005 described culturing of hES cells on pathogen-free human placental fibroblast feeders, and in serum-free conditions, Lee et al. 2005 disclosed culturing of hES cells on mitotically inactivated human uterine endometrial cells, and Richards et al. 2002 described culturing of hES cells on human foetal and adult fibroblast feeders. Furthermore, to avoid contact and mixing between pPS cells, e.g., hES cells, and feeder cells, e.g., mouse EF cells or other non-pPS cells, the pPS may be propagated in the absence of a feeder cell layer. For example, Xu et al. 2001 (Nature Biotechnology 19: 971 ) discloses culturing of pPS cells in the absence of feeder cells, wherein the pPS cells are cultured on an extracellular matrix in a medium conditioned by MEF cells, WO 2001/51616 discloses culturing of pPS cells in the absence of feeder cells, wherein the pPS cells are cultured on an extracellular matrix, e.g., Matrigel ® Basement Membrane Matrix (e.g., BD Biosciences) or laminin, and in a medium conditioned by primary or permanent cell lines, e.g., primary embryonic fibroblasts, telomerised fibroblasts, and fibroblast cells differentiated and selected from cultured pPS cells, and WO 2003/020920 and WO 2006/017370 disclose feeder-free culturing of pPS cells on support structures, such as extracellular matrix, in media supplemented with sufficient fibroblast growth factor.

The pPS cells may be plated typically at >15,000 cells/cm² (optimally 90,000 cells/cm² to 170,000 cells/cm²). Typically, enzymatic dispersion or mechanical dissociation of the cells is halted before cells become completely dispersed (e.g., 5 to 20 min with collagenase type IV). Clumps of -10-2000 may be optimally used to re-plate directly onto the substrate without further dispersal.

The methods of the present invention involve plating of undifferentiated pPS cells onto a substrate which allows adherence of cells thereto. The term "plating" as used herein is synonymous to seeding or inoculating, and in general refers to introducing a cell population into an in vitro environment capable of promoting the survival and/or growth of the introduced cells. Typically, the said environment may be provided in a system which is suitably delimited from the surroundings, such that it can prevent an undesired exchange of matter between the said environment and the surroundings (thereby avoiding, e.g., contamination of the environment or escape of culture medium or cells therefrom), while it can allow for continuous or intermittent exchange of other, useful, matter components between the said environment and the surroundings (e.g., an occasional exchange of a part or all of the culture medium, the continuous exchange of gases, or the harvesting of the cells at the end of culturing, etc.). Usually, environments suitable for culturing of cells can be generated in culture vessels well-known in the art, such as, e.g., cell culture flasks, well plates and dishes of various formats. The said environment comprises at least a medium, in the methods of the invention typically a liquid medium, which supports the survival and/or growth of the cells. The medium may be added to the system before, together with or after the introduction of the cells thereto. The medium may be fresh, i.e., not previously used for culturing of cells, or may comprise at least a portion which has been conditioned by prior culturing of cells therein, e.g., culturing of the cells which are being plated or antecedents thereof, or culturing of cells more distantly related to or unrelated to the cells being plated. pPS cells cultivated without differentiation typically form colonies which attach to a substrate on which the cells propagate. To facilitate plating of so-grown pPS cells according to the invention, such cells or colonies thereof, may be detached from the said substrate and the cells may be at least in part detached from each other, and obtained in a format suitable for subsequent plating. Usually, this format may involve a dispersion of the pPS cells and/or clumps or clusters thereof in a liquid phase consisting of or comprising an isotonic buffer (e.g., phosphate buffered saline) and/or a suitable culture medium (e.g., a medium in which the pPS cells are to be cultured following the plating). Appropriate ways of detaching and dispersing (disassociating) adhering cells are generally known in the art and may be used in the present invention. These involve, e.g., treatment with proteolytic enzymes, chelation of bivalent ions, mechanical disintegration, or combinations of any of the above.

For example, treatment with proteolytic enzymes may result in digestion of proteins which mediate adherence of cells to culture surfaces or substrates, digestion of proteins which mediate cell-cell adherence and/or digestion of protein components of substrates to which the cells attach. Suitable proteolytic enzymes include, but are not limited to, trypsin, collagenase, e.g., collagenase type I, collagenase type II, collagenase type III, collagenase type IV, elastase, Accutase TM (Innovative Cell Technologies), dispase, pronase, papain, plasmin or plasminogen (WO 1994/03586). Concentrations and conditions (e.g., duration, temperature, etc.) at which such proteolytic enzymes are typically used to effect detachment of cells are generally known in the art. For example, collagenase may be typically employed at between 10U/ml to 1000U/ml, e.g., usually at about 200U/ml, or typically at between 0.05mg/ml to 10mg/ml, e.g., usually at about 1-2mg/ml, typically at 37⁰C. For example, trypsin may be typically employed at between 0.005% to 2.5% (w/v), e.g., usually at about 0.05% to 0.25% (w/v), typically at 37⁰C. Typically, cells may be exposed to the proteolytic enzyme dissolved in an isotonic buffer, such as, e.g., PBS or Hanks balanced salt solution. The volume of the enzyme solution for treating the cells may be usefully kept to minimum (e.g., by rinsing the cells in the enzyme solution and decanting excess thereof), such that the enzyme may be suitably diluted after digestion by addition of an excess volume of medium. One skilled in the art is able to monitor the progress of cell detachment and dissociation visually, e.g., macroscopically or using a microscope, and adapt conditions, e.g., amount of enzyme, duration of treatment, etc., accordingly.

For example, chelation of bivalent ions in the cell surroundings, primarily chelation of Ca²⁺ and Mg²⁺ ions, which are believed to contribute to cell adhesion, can be effected using suitable chelators, including, but not limited to EDTA (ethylenediamine tetraacetic acid) or salts thereof, e.g., a disodium salt thereof, and EGTA (ethyleneglycerol tetraacetic acid) or suitable salt thereof, e.g., a disodium salt thereof. For example, EDTA may be typically employed at between 0.05mM and 25mM, e.g., usually at about 0.5mM, typically at 37⁰C. Typically, the cells may be exposed to a bivalent ion chelator dissolved in an isotonic buffer, such as, e.g., PBS without Ca²⁺ and Mg²⁺ ions. Treatment with bivalent ion chelators is usually less destructive to a cell's components (e.g., surface proteins) than treatment with proteases, and cells may be exposed to such chelators for longer periods than to proteases.

For example, mechanical dissociation of cells may involve repeated passing of cell colonies, cell clumps or clusters through a small bore pipette (e.g., a 1000μl micropipette tip) to effect shearing forces dissociating the cells. Mechanical cell dissociation may, when used in isolation, lead to cell damage and may thus be advantageously combined with a prior treatment with proteolytic enzymes and/or chelators. Suitable methods of detaching cells may involve combinations of the above treatments. For example, the cells may be simultaneously treated with a proteolytic enzyme and a chelator. Illustrative but non-limiting formulations are known in the art, e.g., comprising trypsin, e.g., 0.05% (w/v), and EDTA, e.g., 0.5mM or collagenase and EDTA. In addition, cells treated with one or more of proteolytic enzyme and/or chelator may be subsequently mechanically dissociated, e.g., by repeated pipetting through a small bore pipette, to obtain individual cells and/or cell clumps or clusters of a preferred size.

Typically, a suitable method of cell detachment and dispersion should preserve viability of the cells. Preferably, a cell suspension obtained following detachment and dispersion may comprise at least 60% of viable cells, e.g., at least 70%, more preferably at least 80%, and most preferably at least 90% and up to 100% of viable cells. A skilled person will know that cell types may display varying susceptibility to the above treatments or combinations thereof, and will be in general able to choose conditions which ensure a desired degree of cell detachment and dispersion, while preserving cell viability. While the above techniques of detachment and dispersion of adhering cells can be employed generally in cell culture, a skilled person will appreciate that pPS cells tend to be fragile and may require relatively gentle methods. By means of a preferred but non-limiting example, pPS cell colonies can be detached from a culture surface, e.g., gelatine culture substrate, using a step comprising exposing the cells to collagenase, e.g., collagenase type IV. Usually, the said collagenase treatment can also be followed by gently washing the so detached cells in a medium or an isotonic buffer (e.g., PBS), thereby leading to further dispersion of the cells and disassociation of the colonies.

In the present invention, the detachment and dissociation of pPS cells for subsequent plating preferably yields a cell suspension comprising individual pPS cells and/or clumps or clusters of pPS cells. In a non-limiting example, the conditions and parameters of the detachment and dissociation process may be such (as could be easily optimised by a skilled person) as to yield a cell suspension comprising at least 10%, e.g., at least 20%; at least 30%, e.g., at least 40%; at least 50%, e.g., at least 60%; at least 70%, e.g., at least 80%; or at least 90% of individual pPS cells. In another non-limiting example, the said conditions and parameters may be such as to yield a cell suspension comprising at least 10%, e.g., at least 20%; at least 30%, e.g., at least 40%; at least 50%, e.g., at least 60%; at least 70%, e.g., at least 80%; or at least 90% of cells in clumps or clusters of pPS cells. Typically, when gently detaching and dispersing pPS cells for plating according to the invention, a greater portion of the cells may be present in cell clumps or clusters than as individual cells. Suitably, the said pPS cell clumps or clusters may contain, on average, between more than 1 and 1000 cells, between 2 and 800 cells, between 5 and 500 cells, between 10 and 200, between 10 and 150 cells, or between 10 and 100 cells, or between 10 and 200, between 200 and 400, between 400 and 600, between 600 and 800, or between 800 and 1000 cells.

As appreciated by those skilled in the art, detached and dissociated pPS cells may be counted in order to facilitate subsequent plating of the cells at a desired density. Where, as in the present invention, the cells after plating may primarily adhere to a substrate surface present in the culture system (e.g., in a culture vessel), the plating density may be expressed as number of cells plated per mm² or cm² of the said substrate surface. In the present invention, the plating density of undifferentiated pPS cells may lie between about 1x10¹ and 1x10⁶ cells/mm², preferably between about 1x10¹ and 1x10⁵ cells/mm², e.g., between 1x10¹ and 1x10⁴ cells/mm², and more preferably between 1x10² and 1x10⁴ cells/mm², such as, e.g., about 1x10², about 5x10², about 1x10³, about 5x10³, or about 1x10⁴ cells/mm².

In the present invention, undifferentiated pPS cells are plated onto a substrate which allows for adherence of cells thereto, i.e., a surface which is not generally repulsive to cell adhesion or attachment. This may be carried out, e.g., by plating the pPS cells in a culture system (e.g., a culture vessel) which displays one or more substrate surfaces compatible with cell adhesion. When the said one or more substrate surfaces contact the suspension of cells (e.g., suspension in a medium) introduced into the culture system, cell adhesion between the cells and the substrate surfaces may ensue. Accordingly, the term "plating onto a substrate which allows adherence of cells thereto" refers to introducing cells into a culture system which features at least one substrate surface that is generally compatible with adherence of cells thereto, such that the plated cells can contact the said substrate surface. General principles of maintaining adherent cell cultures are well-known in the art.

In general, a substrate which allows adherence of cells thereto may be any substantially hydrophilic substrate. As known in the art, culture vessels, e.g., culture flasks, well plates, dishes, or the like, may be usually made of a large variety of polymeric materials, including, but not limited to polyacrylates, polymethylacrylates, polycarbonates, polystyrenes, polysulphones, polyhydroxyacids, polyanhydrides, polyorthoesters, polyphosphazenes, polyphosphates, polyesters, nylons or mixtures thereof, etc. Generally, culture vessels made of such materials are surface treated after moulding in order to provide for hydrophilic substrate surfaces and thereby enhance the likelihood of effective cell attachment. Surface treatment may take the form of a surface coating, or may involve the use of directed energy at the surface with the intention of generating chemical groups on the polymer surface. These chemical groups will have a general affinity for water or otherwise exhibit sufficient polarity to permit stable adsorption to another polar group. These functional groups lead to hydrophilicity and or an increase in surface oxygen and are properties recognized to enhance cell growth on so modified substrate surfaces. Such chemical groups may include groups such as amines, amides, carbonyls, carboxylates, esters, hydroxyls, sulfhydryls and the like. Examples of directed energy include atmospheric corona discharge, radio frequency (RF) vacuum plasma treatment, and DC glow discharge or plasma treatment (e.g., US 6,617,152). Current standard practices for growing adherent cells may involve the use of defined chemical media with addition of bovine or other animal serum. The added serum, besides providing nutrients and/or growth promoters, may also promote cell adhesion by coating the treated plastic surface with a layer of matrix to which cells can better adhere.

An alternative substrate surface compatible with cell adhesion may be glass, optionally surface treated to introduce functional groups, e.g., as listed above, to increase the hydrophilicity thereof.

Other adherent substrate surfaces may be generated via surface coating, e.g., coating of the polymeric or treated polymeric surfaces as above. In a non-limiting example, the coating may involve suitable poly-cations, such as, e.g., poly-omithine or poly-lysine.

In another example, preferred coating, and accordingly the substrate, comprises one or more components of extracellular matrix, e.g., the ECM proteins fibrin, laminin, collagen, preferably collagen type 1 , gelatine, glycosaminoglycans, e.g., heparin or heparan sulphate, fibronectin, vitronectin, elastin, tenascin, aggrecan, agrin, bone sialoprotein, cartilage matrix protein, fibrinogen, fibulin, mucins, entactin, osteopontin, plasminogen, restrictin, serglycin, SPARC/osteonectin, versican, thrombospondin 1 , or cell adhesion molecules including cadherins, connexins, selectins, by themselves or in various combinations.

Preferred examples may include fibrin, laminin or collagen. Further preferred examples may involve compositions comprising ECM components, such as, e.g., Matrigel ® Basement

Membrane Matrix (BD Biosciences), which is solubilised basement membrane preparation extracted from EHS mouse sarcoma, a tumour rich in ECM proteins, with laminin as a major component, followed by collagen type 4, heparan sulphate proteoglycans, and entactin.

A particularly preferred example of substrate surface for use in the present invention comprises or consists of gelatine. The term "gelatine" as used herein refers to a heterogeneous mixture of water-soluble proteins of high average molecular weight derived from the collagen-containing parts of animals, such as skin, bone and ossein by hydrolytic action, usually either acid hydrolysis or alkaline hydrolysis. The term "gelatine" also encompasses suitable chemical derivatives thereof such as acetylated gelatine or cross- linked gelatine. Suitable protocols for surface treatment of cell culture equipment with gelatine are well-established in the art. By means of illustration and not limitation, culture vessels may be treated for 2 hours or longer, e.g., for 24 hours, with 0.02%-1 % (w/v), typically with about 0.1 % (w/v) gelatine in, e.g., distilled and preferably sterilised water, e.g., autoclave-sterilised.

The above described action, whereby a cell population (here above, undifferentiated pPS cells) is removed from a culture system, e.g., culture vessel, and undergoes replating and subculture, is generally known as "passage" or "passaging". For sake of simplicity, the passage performed at this stage of the method, i.e., involving detachment of undifferentiated pPS cells and replating thereof for subsequent differentiation into MPC and/or MSC, preferably into mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic lineage, is herein referred to as "first passage" (or passage 1 ) within the method of the invention.

Typically, after plating of the pPS cells (i.e., following the "first passage"), the cell suspension is left in contact with the adherent surface to allow for adherence of cells from the plated cell population to the said substrate.

In embodiments, the pPS cell suspension may be contacted with the adherent surface for at least about 0.5h, e.g., for at least about 1 h, preferably for at least about 2h, for at least about 4h, more preferably for at least about 8h, e.g., for at least about 12h, even more preferably for at least about 16h, e.g., for at least about 2Oh, and most preferably for at least about 24h or more, e.g., for at least about 28, 32, 36, 40, 44 or 48h.

In other preferred embodiments, the primary cell suspension may be contacted with the adherent surface for between about 2h and about 48h, e.g., for about 12h, about 24h, about 36h or for about 48h. Although longer contacting times (before removal of the non-adherent matter) are possible, they are in general not necessary. In contacting the pPS cells with adherent substrate, the cells may be advantageously suspended in a suitable culture medium as described elsewhere in this specification. Preferably, the medium may have the features (presence of serum or plasma and allowing differentiation of pPS cells) as the medium used in the ensuing step of culturing the attached cells. For example, the composition of the medium may be the same or substantially the same as the composition of medium used in the ensuing step of culturing the attached cells. Otherwise, the compositions of the medium may be different.

After cells from the pPS suspension are allowed to attach to adherent substrate as described above, non-adherent matter is removed from the culture system. Non-adherent matter may comprise, but is not limited to, cells that have not attached to the adherent substrate (such as, e.g., cells which are not prone to adherence, or cells which would not attach within the time allowed therefore), non-viable or dead cells, cell debris, etc. Non-adherent matter can be typically removed by discarding medium from the culture system, whereupon adherent cells remain attached to the substrate, and optionally washing, once or repeatedly, the adherent cells and the culture system with suitable medium or buffer (e.g., PBS). Hereby, cells from the pPS suspension which have adhered to the substrate surface, are selected for further culturing.

After plating of the undifferentiated pPS cells and allowing for adherence thereof to substrate as described above (i.e., following the "first passage" of pPS cells within the method), the cells which have adhered to the said substrate are cultured in a medium comprising serum or plasma and allowing differentiation of the pPS cells. The term "culturing" is common in the art and broadly refers to maintenance and/or growth of cells.

The term "plasma" is as conventionally defined. Plasma is usually obtained from a sample of whole blood, which is provided or contacted with an anticoagulant, such as heparin, citrate (e.g., sodium citrate or acid citrate dextrose), oxalate or EDTA, upon or shortly after drawing the blood sample, to prevent clotting. Subsequently, cellular components of the blood sample are separated from the liquid component (plasma) by an appropriate technique, typically by centrifugation. The term "plasma" therefore refers to a composition which does not form part of a human or animal body. The term "serum" is as conventionally defined. Serum can be usually obtained from a sample of whole blood by first allowing clotting to take place in the sample and subsequently separating the so formed clot and cellular components of the blood sample from the liquid component (serum) by an appropriate technique, typically by centrifugation. Clotting can be facilitated by an inert catalyst, e.g., glass beads or powder. Advantageously, serum can be prepared using serum-separating vessels (SST) known in the art, which contain the inert catalyst to facilitate clotting and further include a gel with density designed to become positioned between the liquid component and the clot and cellular components after centrifugation, thus simplifying separation. Alternatively, serum can be obtained from plasma by removing the anticoagulant and fibrin. The term "serum" hence refers to a composition which does not form part of a human or animal body.

The isolated plasma or serum can be used directly in the methods of the present invention. They can also be appropriately stored for a later use in the method of the present invention. Typically, plasma or serum can be stored for shorter time periods, e.g., up to about 1-2 weeks, at a temperature above the respective freezing points of plasma or serum, but below ambient temperature. Usually, this temperature will be about 15⁰C or less, preferably about 1O⁰C or less, more preferably about 5⁰C or less, e.g., about 5⁰C, 4⁰C, 3⁰C, 2⁰C or about 1⁰C, most preferably about 5⁰C or about 4⁰C. Alternatively, plasma or serum can be stored at below their respective freezing points, i.e., by freeze storage. As usual in the art, advantageous temperatures for freeze storage of plasma or serum can be about -7O⁰C or less, e.g., about -75⁰C less or about -8O⁰C or less. Such temperatures may advantageously prevent any thawing of the stored plasma or serum, thereby preserving the quality thereof. Freeze storage can be used irrespective of the time period for which the plasma or serum need to be stored, but may be particularly suitable if longer storage is required, e.g., for longer than a few days or for longer than 1-2 weeks.

Prior to storage or use, the isolated plasma or serum can be heat inactivated. Heat inactivation is used in the art mainly to remove the complement. Heat inactivation typically involves incubating the plasma or serum at 56⁰C for 30 to 60min, e.g., 30min, with steady mixing, after which the plasma or serum is allowed to gradually cool to ambient temperature. A skilled person will be aware of any common modifications and requirements of the above procedure.

Optionally, the plasma or serum may also be sterilised prior to storage or use. Usual means of sterilisation may involve, e.g., filtration through one or more filters with pore size smaller than 1μm, preferably smaller than 0.5μm, e.g., smaller than 0.45μm, 0.40μm, 0.35μm, 0.30μm or 0.25μm, more preferably 0.2μm or smaller, e.g., 0.15μm or smaller, 0.10μm or smaller.

Suitable sera or plasmas for use in the media of the present invention may include human serum or plasma, or serum or plasma from non-human animals, preferably non-human mammals, such as, e.g., non-human primates (e.g., lemurs, monkeys, apes), foetal or adult bovine, horse, porcine, lamb, goat, dog, rabbit, mouse or rat serum or plasma, etc.

In an embodiment, the serum or plasma may be obtained from an organism of the same species as is the species of the pPS cells that are subjected to the present methods. In a non-limiting example, human serum or plasma may be used for culturing human pluripotent stem cells (e.g., hES, hEG).

In a preferred embodiment, the said medium comprises bovine serum or plasma, preferably foetal bovine (calf) serum or plasma, more preferably foetal bovine (calf) serum (FCS or FBS).

In another embodiment, the medium used to culture human pluripotent cells, such as hES or hEG cells, comprises bovine serum or plasma, preferably foetal bovine (calf) serum or plasma, more preferably foetal bovine (calf) serum (FCS or FBS).

In another embodiment, the invention foresees the use of any combination of the above plasmas and/or sera.

In embodiments, the medium may comprise between about 0.5% and about 40% (v/v) of serum or plasma, preferably between about 5% and 40% (v/v), e.g., between about 5% and

30% (v/v), more preferably between about 8% (v/v) and about 25% (v/v), e.g., between about

10% (v/v) and about 20% (v/v), or more than about 15% (v/v), e.g., between about 15% (v/v) and about 25% (v/v), e.g., between about 15% (v/v) and about 20% (v/v), or more than about

18% (v/v), e.g., between about 18% (v/v) and about 25% (v/v), e.g., between about 18% (v/v) and about 20% (v/v) of serum or plasma. Examples include, e.g., about 8% (v/v), about 9%

(v/v), about 10% (v/v), about 11 % (v/v), about 12% (v/v), about 13% (v/v), about 14% (v/v), about 15% (v/v), about 16% (v/v), about 17% (v/v), about 18% (v/v), about 19% (v/v), about

20% (v/v), about 21 % (v/v), about 22% (v/v), about 23% (v/v), about 24% (v/v), or about 25%

(v/v) of serum or plasma. In a particularly preferred embodiment, the medium may comprise about 20% (v/v) of serum or plasma. In an embodiment, the medium will comprise basal medium formulation as generally known in the art. Suitable basal media formulations include, but are not limited to, Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), BGJb, F-12 Nutrient Mixture (Ham), Knock-out DMEM (KO DMEM) and the like, which are commercially available (e.g., Invitrogen, Carlsbad, California), and MSCGM™ mesenchymal stem cell medium available from Cambrex (East Rutherford, New Jersey). Compositions of the above basal media are generally known in the art and it is within the skill of one in the art to modify or modulate concentrations of media and/or media supplements as necessary for the cells cultured. Such basal media formulations contain ingredients necessary for mammal cell development, which are known per se. For example, these ingredients usually include inorganic salts (in particular salts containing Na, K, Mg, Ca, Cl, P, and possibly Cu, Fe, Se and Zn), physiological buffers (e.g., HEPES, bicarbonate), amino acids, vitamins, and sources of carbon (e.g. glucose, pyruvate, e.g., sodium pyruvate), and may also comprise antioxidants (e.g., glutathione), nucleotides, nucleosides and/or nucleic acid bases, ribose, deoxyribose, etc. It will also be apparent that many media are available as low-glucose formulations with or without sodium pyruvate. For example, see US 5,486,359 for the detailed composition of the standard DMEM, enhanced DMEM, BGJb and F-12 Nutrient Mixture (Ham) media. For use in the methods of the invention, these media are supplemented with a useful volume of suitable serum or plasma, as defined above. In addition, the media may be further supplemented with one or more compounds of interest, including, but not limited to, any of sodium bicarbonate, L-glutamine, non-essential amino acids, β-mercaptoethanol and antibiotic and/or antimycotic components, such as, e.g., penicillin, streptomycin and/or amphotericin, etc.

In a particularly preferred embodiment, the basal medium may be Knock-out DMEM (KO DMEM) manufactured by Invitrogen, optionally and preferably supplemented with any or combination or, preferably, all of L-glutamine (e.g., 2mM), non-essential amino acids

(Invitrogen, e.g., 1 %) and β-mercaptoethanol (e.g., 0.1 mM). This medium may be supplemented with serum or plasma as described above, in a preferred embodiment with

20% (v/v) of plasma or serum, and more preferably foetal bovine (calf) serum (e.g., from Hyclone, Logan, UT).

In the present invention, the medium allows differentiation of pPS cells, meaning that the medium does not contain components, in sufficient quantity, which would suppress this differentiation, or would cause proliferation and/or maintenance of pPS in undifferentiated or substantially undifferentiated state. By means of illustration, such components might include, e.g., basic fibroblast growth factor (b-FGF), or conditioned medium of feeder cells (e.g., mouse or human fibroblast cells as described above), in sufficient quantity. Accordingly, the culture system into which the pPS are plated for differentiation into MSC also preferably does not contain feeder cells, which might otherwise counteract pPS differentiation.

In an embodiment, the medium does not contain growth factors and/or differentiation factors exogenous to (i.e., supplemental to or in addition to) such factors contributed to the medium by inclusion of the serum or plasma as defined above in the medium. A skilled person would appreciate that the ordinary components of basal media (before addition of serum or plasma), e.g., in particular, isotonic saline, buffers, inorganic salts, amino acids, carbon sources, vitamins, antioxidants, pH indicators and antibiotics, are not considered growth factors or differentiation factors in the art. On the other hand, serum or plasma is a complex composition possibly comprising one or more such growth factors or differentiation factors. The term "growth factor" or "differentiation factor" as used herein refers to a biologically active substance which influences proliferation, growth, differentiation, survival and/or migration of various cell types, and may effect developmental, morphological and functional changes in cells, tissues or an organism, either alone or when modulated by other substances. A growth factor or differentiation factor may typically act by binding, as a ligand, to a receptor (e.g., surface or intracellular receptor) present in cells responsive to the growth factor. A growth factor or differentiation factor herein may be particularly a proteinaceous entity comprising one or more polypeptide chains, although occasionally simple compounds, e.g., organic compounds, may also show the effect of a growth factors or differentiation factor, e.g., retinoic acid (e.g., in neuronal differentiation), DMSO (e.g., in hepatocyte maturation), etc. By means of example and not limitation, the term "growth factor" or "differentiation factor" encompasses the members of the fibroblast growth factor (FGF) family, bone morphogenic protein (BMP) family, platelet derived growth factor (PDGF) family, transforming growth factor beta (TGF-beta) family, nerve growth factor (NGF) family, the epidermal growth factor (EGF) family, the insulin related growth factor (IGF) family, the hepatocyte growth factor (HGF) family, hematopoietic growth factors (HeGFs), the platelet-derived endothelial cell growth factor (PD-ECGF), angiopoietin, vascular endothelial growth factor (VEGF) family, glucocorticoids, and the like. The term "growth factor" or "differentiation factor" also comprises cytokines. Cytokines are growth factors that have been, historically, associated with the function of hematopoietic and immune systems. Cytokines comprise, by means of example and not limitation, interleukins, lymphokines, monokines, interferons, colony-stimulating factors and chemokines. Particular, but non-limiting, examples of cytokines include interleukins (e.g., IL-1 , IL-2, IL-3, IL-4, IL-5, IL- 6, IL-7, IL-9, IL-10, IL-11 , IL-12a, IL-12b, IL-13), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony, stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), stem cell factor (SCF), interferon-alpha (IFN-alpha), interferon- beta (IFN-beta), interferon-gamma (IFN-gamma), or a hybrid interferon, erythropoietin (EPO), leukaemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin 1 (CT-1 ), growth hormone (GH), pre-B-cell leukaemia transcription factor-1 (PRL), tumour necrosis factor (TNF) family of cytokines (e.g., TNF-alpha and TNF-beta), chemokines, e.g., C chemokines including XCL1 (lymphotactin), CC chemokines including CCL1 (I-309) to CCL28, e.g., MIP1 alpha (CCL3), CXC chemokines such as CXCL1 (GRO alpha) to CXCL14 (BRAK), CX3C chemokines such as CX3CL1 (fractalkine), and the like.

A skilled person will understand that the medium may contain one or more growth factors and/or differentiation factors exogenous to (i.e., supplemental to or in addition to) such factors contributed to the medium by inclusion of the serum or plasma as defined above in the medium, e.g., one or more growth factors and/or differentiation factors listed above. For example, addition of some factors might not influence the differentiation of pPS into MSC, in which case such factors may be added, albeit they would likely have no discernible effect. More interestingly, other exogenously added growth factors or differentiation factors may further stimulate the differentiation process obtained with the methods of the present invention, therefore providing further advantages. In an embodiment, culturing of the pPS cells following the "first passage" as above may be carried out until the cells have become preferably at least 70%, e.g., at least 80%, more preferably at least 90%, e.g., at least 95% or even fully confluent. The term "confluence" as used herein refers to a density of cultured cells in which the cells contact one another covering substantially all of the surfaces available for growth (i.e., fully confluent). In another embodiment, the said culturing may be carried out for at least 10 days, e.g., at least 11 , 12, 13 or 14 days, or preferably for at least 15 days, e.g., at least 16, 17, 18 or 19 days, or more preferably for at least 20 days, e.g., at least 21 , 22, 23 or 24 days. Preferably, the said culturing may be carried out for no longer than 35 days, e.g., for no longer than 30 days.

In an embodiment, the said culturing may be carried out for at least 20 days, e.g., at least 21 , 22, 23 or 24 days, such as for about 24 days. A skilled person will appreciate that prolonged culturing of cells as above may necessitate regular exchange of the medium to ensure suitable environment for maintenance and/or growth of cells (e.g., sufficient nutrients, minerals, optimal pH, removal of decay products, etc.). Such may involve the exchange of substantially all medium for fresh medium or, alternatively, exchange of a certain fraction of the medium for fresh medium (the latter situation preserving a continuous presence of factors synthesised by the cells in the culture). By ways of a non-limiting example, between 1/4 and 3/4 of the medium, e.g., about 1/4 or about 1/3, or about 1/2 or about 2/3 or about 3/4 or even more of the medium may be exchanged for fresh medium.

A skilled person will further appreciate that other conditions will be preferably maintained for the said culturing, concerning the temperature of cultivation, e.g., between 30 and 37⁰C, such as about 36.5⁰C, CO₂ atmosphere, e.g., from 0.1 to 10% (v/v), humidity, e.g., about 90-100%, or the like, generally as known in the art for mammalian cell culture.

Following the above culturing the pPS cells (e.g., after the specified duration thereof and/or after the cultured cells have reached the specified confluence), the cells are passaged at least once. For sake of simplicity, the passage performed at this stage of the method, (i.e., the passage directly subsequent to plating of undifferentiated pPS cells onto an adherent substrate and culturing the adhering cells in a medium comprising plasma or serum and allowing differentiation of the pPS cells, as described above) is herein referred to as "second passage" (or passage 2) within the method of the invention. The cells may be passaged at least one time and preferably two or more times. Each passage subsequent to passage 2 is referred to herein with a number increasing by 1 , e.g., passage 3, 4, 5, 6, etc.

When passaged (i.e., passage 2 and subsequent), the cultured cells are detached and dissociated from the culture substrate and from each other. Detachment and dissociation of the cells can be carried out as explained above, e.g., by enzymatic treatment, treatment with bivalent ion chelators or mechanical treatment, or any combination thereof. Preferably, the detachment and dissociation of the cultured cells would yield a substantial proportion of the cells as single cells. For example, 40% or more of the cells can be recovered as single cells, e.g., at least 50%, preferably at least 60%, e.g., at least 70%, more preferably at least 80%, e.g., at least 90% or at least 95% of the cells may be recovered as single cells. Moreover, the remaining cells may be present in cell clumps or clusters the majority of which can contain a relatively small number of cells, e.g., on average, between more than 1 and 10 cells, e.g., less than 8 cells, preferably less than 6 cells, more preferably less than 4 cells, e.g., less than 3 or less than 2 cells.

Preferably, the said cell detachment and dissociation may involve enzymatic digestion, favourably using trypsin (e.g., as described above), optionally and preferably in combination with chelation of bivalent ions, favourably using EDTA (e.g., as described above), and followed by mechanical dissociation of the so-treated cells. The latter may involve, e.g., repeated passing of the cells through a small bore pipette (e.g., a 1000μl micropipette tip) and/or pipetting out a stream of a suspension containing the cells against a solid surface (e.g., against the wall of the culture vessel).

The present inventors have observed that some cultures of the pPS cells after the first passage may contain rather large clumps of cells, which may not come loose from the culture substrate upon usual detachment paradigm. The inventors have realised that the method of the invention is not affected if such cell clumps remain in the plate and are not a subject to detachment and re-plating as described herein. Accordingly, in an embodiment, such cell clumps may not be detached. Next, the so detached and dissociated cells (typically as a cell suspension in an isotonic buffer or a medium) are re-plated onto a substrate which allows for adherence of cells thereto, and are subsequently cultured in a medium comprising serum or plasma and allowing for further differentiation of the said cells.

Typically, the cells may be re-plated at plating density of between 1x10² and 1x10⁶ cells/mm², and preferably between 1x10³ and 1x10⁵ cells/mm², e.g., at about 1x10³ cells/mm², at about 5x10³ cells/mm², at about 1x10⁴ cells/mm², at about 5x10⁴ cells/mm², or at about 1x10⁵ cells/mm².

Alternatively, the cells may be re-plated at a splitting ratio of, e.g., between about 1/8 and 1/2, preferably between about 1/4 and 1/2, and more preferably at about 1/2 or about 1/3. The splitting ratio denotes the fraction of the passaged cells which is seeded into an empty (typically a new) culture vessel of the same surface area as the vessel from which the cells were obtained. The adherent substrate onto which the cells are re-plated is as described in detail elsewhere in this specification. The substrate may be of the same kind as the substrate onto which the pPS cells were plated in the previous step of the method (i.e., upon first passage), including preferred embodiments of such substrate described above, or may be different. Preferably, this substrate is also gelatine, as described above. In another preferred embodiment, the substrate may be plastic.

Further, the culture medium in which the cells are subsequently cultured is as described elsewhere in the specification. The medium may be of the same composition as the medium in which the pPS cells were cultured in the previous step of the method (i.e., following the first passage), including preferred embodiments described above (such as, without limitation, e.g., preferred embodiments relating to the origin and amount of serum or plasma in the medium), or may be different. In a preferred embodiment, the medium may have the same composition. In a preferred, though non-limiting example, the medium may be the basal medium Knock-out DMEM (KO DMEM) manufactured by Invitrogen, optionally and preferably supplemented with any or combination of or, preferably, all L-glutamine (e.g., 2mM), non-essential amino acids (Invitrogen, e.g., 1 %) and β-mercaptoethanol (e.g., 0.1 mM). This medium may be supplemented with serum or plasma, preferably serum, as described above, including preferred embodiments described above, in a preferred exemplary embodiment with 20% (v/v) of plasma or serum, preferably serum, and more preferably foetal bovine (calf) serum (e.g., from Hyclone, Logan, UT). The medium may be fresh or may contain a fraction of conditioned medium recovered from the cells before passaging.

The so passaged cells are further cultured as defined above, advantageously until the cells have become at least 50% confluent, e.g., at least 60%, preferably at least 70%, e.g., at least 80%, more preferably at least 90%, e.g., at least 95% or even fully confluent. The present inventors have realised that the cell population obtained at this stage of the method (i.e., following culturing of the cells after second passage as defined herein) comprises a considerable fraction of cells having features of MPC and/or MSC, and preferably of mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway, in particular morphological characteristics thereof. Advantageously, the present inventors further realised that if the cell population obtained at this stage is subjected to at least one additional passage, and possibly more than one passages, the proportion of MPC and/or MSC, and preferably of the mesenchymal progenitors capable of and/or committed towards differentiation along the osteogenic pathway in the cell population, e.g., as judged by morphology, is increased and even a substantially homogeneous population of the respective cells may be obtained. For sake of simplicity, the said additional passages refer to the third and each subsequent passage as defined above. Hence, according to the invention, following the first passage as defined herein, the cells are passaged at least once (i.e., second passage) and preferably two (i.e., second and third passage) or more times (i.e., second, third and each subsequent passage). For example, the cells may be passaged at least 2 times, at least 3 times, at least 4 times or at least 5 times following the first passage. In another embodiment, the cells may be passaged between 2 and 10 times, e.g., between 2 and 8 times, or between 2 and 5 times, following the first passage. The additional passages (e.g., cell detachment and dispersion, replating, substrate, etc.) and culturing (e.g., medium, medium changes, resulting confluence, etc.) may be performed at conditions substantially identical or analogous to those of the second passage, as described above, including preferred embodiments thereof and may include modifications which would be obvious to the skilled person.

The present inventors have realised that the cell population obtained upon culturing of the cells following third passage and following each further passage comprises a considerable fraction of cells having features of MPC and/or MSC, in particular the morphological MPC and/or MSC characteristics, and/or cells having reactivity with MSC specific marker molecules, e.g., staining for one, two, three or more of the following markers: CD73 (SH3, SH4), CD106 (VCAM), CD166 (ALCAM), CD90, CD105 (SH2), CD29, CD44, CD54, GATA-4, STRO-1 and alkaline phosphatase, while being negative for hematopoietic lineage cell markers (e.g., CD14, CD 34 and CD45). The so obtained population may be substantially homogeneous MPC and/or MSC population, preferably substantially homogeneous population of mesenchymal progenitor cells capable of and/or committed toward differentiation along the osteogenic pathway.

In an embodiment, the MPC and/or MSC cells comprised in the said population are capable of being differentiated into cells of at least the osteogenic lineage, e.g., assayed as known in the art, i.e., are mesenchymal progenitor cells capable of differentiation along the osteogenic pathway.

In a further embodiment, the MPC and/or MSC cells cannot be differentiated into cells of the chondrogenic and/or adipogenic lineages. In a preferred embodiment, the mesenchymal progenitor cells can be differentiated into cells of (at least) the osteogenic lineage, but not to cells of the chondrogenic and/or adipogenic lineages.

In a preferred embodiment, the MPC and/or MSC are thus mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway.

In a particularly preferred embodiment, the mesenchymal progenitor cells are thus committed towards differentiation along the osteogenic pathway.

For example, such mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway may express one or more markers of osteoblastic differentiation, e.g., one or more markers of an early stage of osteoblastic differentiation, such as, for example, CBFA1/RUNX2 and/or collagen type I).

As noted above, the method of the invention, when including the said third (or optionally further) passage provides for a cell population with a considerable proportion of MPC and/or MSC, preferably of mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway, as defined, e.g., by differentiation potential, cell morphology and/or displayed marker molecules. Typically, such population will comprise at least about 5% of the respective cells ( MPC and/or MSC, or preferably of mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway), e.g., at least about 10%, but the inventors found that typically higher proportions of the respective cells will be obtained, e.g., at least 20%, at least 30%, at least 40% or more, or at least 50%. Moreover, as shown in the examples, the said method may in fact yield even higher proportion of the respective cells, such as at least 60%, at least 70%, at least 80%, at least 90%, or even a substantially homogeneous population can be obtained as defined herein. In particularly noteworthy experiments, essentially all cells displayed one or more markers typical of MPC and/or MSC, ore preferably of mesenchymal progenitor cells capable of and/or committed towards differentiation along the osteogenic pathway.

A skilled person will appreciate that the cell population obtained by the present method, which comprises MPC and/or MSC and/or which is a substantially homologous MPC and/or MSC population, may be harvested (e.g., by suitable detachment technique) and optionally further enriched for cells displaying specific MPC and/or MSC characteristics by methods generally known in the art (hence, such cells can be isolated from the said population). By means of illustration and not limitation, cells displaying one or more surface molecules characteristic of MPC and/or MSC, e.g., one or more of the markers listed above, may be recognised by specific (labelled) antibodies or other recognition agents against such as markers and sorted out from cells not displaying such surface molecules, e.g., using fluorescence activated cell sorting (see, e.g., Barberi et al. 2005 for selection of CD73+ MSC cells) or using affinity binding to, e.g., columns, beads or surfaces (panning). Any other ways of enrichment for MPC and/or MSC cells are also included in the invention. The term "antibody" refers to both polyclonal and monoclonal antibody encompasses not only intact immunoglobulin molecules, but also such fragments and derivatives of immunoglobulin molecules (such as single chain Fv constructs, diabodies, and fusion constructs) as may be prepared by techniques known in the art, and retaining a desired antibody binding specificity.

When a population comprising MPC and/or MSC and/or a substantially homogeneous MPC and/or MSC population is achieved obtained by the methods of the invention, and possibly further enriched for MPC and/or MSC, the said cell population may next be maintained and/or propagated in conditions which allow for MPC and/or MSC growth and doubling without differentiation. Propagation of MPC and/or MSC in undifferentiated state can advantageously increase the number of MPC and/or MSC available for obtaining further differentiated cell phenotypes therefrom. The so obtained cell populations comprising MPC and/or MSC and/or a substantially homogeneous MPC and/or MSC can be cryopreserved for further use, as generally known in the art for mammalian cells, and for MPC and/or MSC in particular. For example, MPC and/or MSC can be propagated in such conditions for up to about at least 20 passages, e.g., up to about 15 passages, up to about 10 passages, or up to about 5 passages, e.g., for 5, 4, 3, 2 or 1 passage. The passage number also determines for how many population doublings a given cell population of MPC and/or MSC would be propagated in undifferentiated state. For example, an MPC and/or MSC cell population can be so propagated for up to about 40 population doublings, e.g., up to about 30, up to about 20, up to about 10 or up to about 5 population doublings. A moderate number of passages or population doublings may be preferred to maintain viability and differentiation potential of the propagated MPC and/or MSC. A skilled person would be able to determine a suitable balance between expansion of MPC and/or MSC and sustaining a satisfactory viability and differentiation potential thereof. Any conditions suitable for propagation of MPC and/or MSC in undifferentiated state may be employed, such as disclosed, e.g., in US Pat. No. 5,486,359; US Pat. No. 5,811 ,094; US Pat. No. 5,736,396; US Pat. No. 5,837,539; or US Pat. No. 5,827,740, Pittenger et al. 1999, Zuk et al. 2001 , Young et al. 2001 , or Barberi et al. 2005. In an embodiment, to increase the replicative capacity of the obtained MPC and/or MSC cells, these cells can be telomerised. A cell is described as "telomerised" if it has been genetically altered with a nucleic acid encoding a telomerase reverse transcriptase (TERT) of any species in such a manner that the TERT is transcribed and translated in the cell. The term also applies to progeny of the originally altered cell that have inherited the ability to express the TERT encoding region at an elevated level. The TERT encoding sequence is typically taken or adapted from a mammalian TERT gene, exemplified by human and mouse TERT, as indicated below.

Cells may be telomerised by genetically altering them with a suitable vector, so that they express the telomerase catalytic component (TERT) at an elevated level. Particularly suitable is the catalytic component of human telomerase (hTERT), provided in WO 1998/14592. For some applications, other TERT sequences can be used.

Typically, the vector will comprise a TERT encoding region under control of a heterologous promoter that will promote transcription in the cell line. For example, sequences that can drive expression of the TERT coding region include viral LTRs, enhancers, and promoters (such as MPSV, SV40, MoLV, CMV, MSCV, HSV TK), eukaryotic promoters (such as β-actin, ubiquitin, EF1a, PGK) or combinations thereof (for example, the CMV enhancer combined with the β-actin promoter). Expression of a marker gene can be driven by the same promoter as the TERT gene, either as a separate expression cassette, as part of a polycistronic transcript (in which the coding regions of TERT and the marker gene are separated by an IRES sequence, allowing both individual proteins to be made from a single transcript driven by a single promoter), or as part of the same cassette (a fusion between the coding regions of both TERT and the marker gene, producing a protein that provides the functions of both TERT and the marker gene). Transfection and expression of telomerase in human cells is described in Bodnar et al. (Science 279:349, 1998) and Jiang et al. (Nat. Genet 21 : 111 , 1999). Before and after telomerisation, telomerase activity and hTERT expression can be determined using standard reagents and methods. For example, pPS cells are evaluated for telomerase using TRAP activity assay (Kim et al. 1997. Science 266: 2011 ; Weinrich et al. 1997. Nature Genetics 17: 498; Reubinoff et al. 2000). hTERT expression can also be evaluated by RT-PCR.

Other methods of immortalizing cells are also contemplated, such as genetically altering the cells with DNA encoding the SV40 large T antigen (US 5,869,243, WO 1997/32972), infecting with Epstein Bar Virus, introducing oncogenes such as myc and ras, introducing viral replication genes such as adenovirus E1a, and fusing cells having the desired phenotype with an immortalized cell line. Transfection with oncogenes or oncovirus products is usually less suitable when the cells are to be used for therapeutic purposes. In general, it might be preferred that MPC and/or MSC are not modified by telomerisation or other ways of immortalisation when the use of the MSC or progeny thereof, including differentiated progeny thereof, is contemplated in therapy, e.g., where such cells are to be introduced to human or animal, esp. human, body.

The MPC and/or MSC or progeny thereof, including differentiated progeny, e.g., cell types of various tissues of mesodermal origin, may in an aspect of the invention be intended for therapeutic applications, e.g., for tissue engineering and cell therapy.

Where administration of such cells to a patient is contemplated, it may be preferable that the pPS cells, e.g., hES or hEG, subjected to the method of the present invention to obtain MPC and/or MSC populations, are selected such as to maximise, at least within achievable limits, the tissue compatibility between the patient and the administered cells, thereby reducing the chance of rejection of the administered cells by patient's immune system (e.g., graft vs. host rejection).

The ability of the immune system to differentiate self from non-self is to a large extent determined by products of the major histocompatibility complex (MHC), whose genes are on chromosome 6 and belong to the immunoglobin gene super-family. Class I MHC products consist of HLA-A, HLA-B and HLA-C; these have a wide distribution and are present on the surface of essentially all nucleated cells and on platelets. Class Il MHC products consist of HLA-D, HLA-DR, HLA-DP, and HLA-DQ; they have a more limited distribution, including on B cells, macrophages, dendritic cells, Langerhans' cells, and activated (but not resting) T cells. The HLA loci are generally multi-allelic, e.g., using specific antibodies, at least 26 HLA-A alleles, 59 HLA-B alleles, 10 HLA-C alleles, 26 HLA-D alleles, 22 HLA-DR alleles, nine HLA- DQ alleles and six HLA-DP alleles can be recognized. Because HLA loci are closely linked, the HLA antigens may also be present as conserved haplotypes.

A subject in need of therapy with cells of the present invention may be screened for the presence of anti-HLA antibodies and for his HLA genotype and/or phenotype (e.g., on lymphocytes; e.g., using serological methods or genetic DNA analysis). pPS cells or cell lines, esp. ES cells or cell lines, may be typically tested for their HLA phenotype and/or genotype pPS cells may be selected for production of the MPC and/or MSC or other derived cells for administration, which have either identical HLA haplotypes to the patient, or which have the most HLA antigen alleles common to the patient and none or the least of HLA antigens to which the patient contains pre-existing anti-HLA antibodies. The probability that the transplanted cells will be successfully accepted increases with the number of identical HLA antigens. A skilled person will understand the further variations of these considerations.

Other ways of obtaining MHC profile resembling the patient's are also contemplated, e.g., genetic manipulation of the obtained MPC and/or MSC cells or progeny thereof. In the present invention, the pPS cells or cell lines, e.g., ES cells or cell lines, or preferably, the MPC and/or MSC cells or populations comprising MPC and/or MSC derived from the pPS cells or cell lines according to the invention, as well the progeny and derivatives thereof, including (partly) differentiated progeny or derivatives of the MPC and/or MSC cells, may be stably or transiently transfected or transformed with a nucleic acid of interest prior to further use, e.g., in therapy or research. Nucleic acid sequences of interest may include, but are not limited to, e.g., those encoding gene products which enhance the growth, differentiation and/or functioning of cell types useful in therapy, e.g., cell types of various mesenchymal lineages, such as, e.g., the osteocytic (bone), chondrocytic (cartilage), myocytic (muscle), tendonocytic (tendon), fibroblastic (connective tissue), adipocytic (fat) or stromogenic (marrow stroma) cell lineages, preferably osteocytic (bone) lineage, or to deliver a therapeutic gene to a site of administration of such cells. A vector is designed using the known encoding sequence for the desired gene, operatively linked to a promoter that is either pan-specific or specifically active in the differentiated cell type. Of particular interest are gene products involved in formation and/or regeneration of particular connective tissues. A non-limiting example is, e.g., expression of BMP-2 or BMP 4 to aid bone tissue formation and/or regeneration or MyoD for muscle differentiation.

In a preferred embodiment, the pPS cells, are not contacted prior to and/or during the method of the present invention with any component obtained from a non-human animal, in particular non-human mammal. If cells of the present invention are to be administered to a human subject, the absence of contact between the cells and components obtained from non-human animals ensures optimal acceptance of the cells by the subject and avoids accidental transmission of infectious agents thereto. The latter concern becomes increasingly important due to the appearance of prion diseases, e.g., BSE, which can be transmitted from animals to humans. Derivation and maintenance of human pPS cells or cell lines in conditions not including non-human animal components (e.g., derivation on human feeders and/or in a medium conditioned with human feeders) is known in the art. In a particular embodiment, the human pPS cells are not contacted with any serum component derived from a non-human animal. For example, the present method may use human plasma or serum instead of non-human animal sera. E.g., in an embodiment the present method does not use FCS or other non-human animal serum components. In an embodiment, the serum or plasma may be derived from the patient to whom the cells of the present invention or derivatives thereof are to be administered. In another embodiment, the said serum or plasma may be at least partly derived from one or more other human subjects. Such serum or plasma may be tested for the absence of pathogens, e.g., common pathogens, such as HIV, hepatitis virus, etc., as known in the art.

Under appropriate conditions, MPC and/or MSC can differentiate further into one or more adult connective tissue cell types, such as, e.g., fibroblasts, chondroblasts, osteoblasts, odontoblasts, reticular cells or adipocytes. In a particularly preferred embodiment, the mesenchymal progenitor cells of the invention that are capable or and/or committed towards differentiation into cells of osteogenic lineage, can be differentiated to such cells. Suitable paradigms and conditions for inducing differentiation of MSC cells have been referred to in the present disclosure and are described, e.g., in Pittenger et al. 1999, Zuk et al. 2001 , Young et al. 2001 , Wakitani et al. 1995, Gang et al. 2004, Barberi et al. 2005, and the elsewhere cited patent documents.

Preferably, the mesenchymal progenitor cells can differentiate into cells of the osteogenic lineage, e.g., osteoblasts and/or osteocytes. Osteoblasts and bone precursor cells may typically have at least one characteristic, and may display at least two, at least three, at least four or at least five characteristics, from the following list: (a) density between -1.050 and -1.090 g/cm3; (b) positive for osteonectin (positive in osteoblasts and precursors); (c) positive for osteocalcin (specific for mature osteoblasts); (d) a cell diameter between -6 to ~70μm ; (e) substantially cuboidal shape; (f) upregulated production alkaline phosphatase (ALP) (more specifically, ALP of the bone-liver- kidney type); (g) positive for type I collagen (procollagen) and/or for vimentin; (h) positive for other osteoblast-specific markers, such as BMP receptors, PTH receptors, or CD105 (endoglin); (i) evidence of ability to mineralize the external surroundings, or synthesize calcium-containing extracellular matrix, when exposed to osteogenic or mineralization medium (Jaiswal et al. 1997. J Cell Biochem 64: 295-312), e.g., by Kossa staining.

The skilled reader will know that chondrocytes typically express type Il collagen, aggrecan, or proteoglycans that stain with alcian blue. In the mature form, chondrocytes will be less than 1 % positive for elastin, type I collagen, type X collagen, or osteocalcin. Cardiomyocytes and their precursors typically express cardiac troponin I (cTnl), cardiac troponin T (cTnT), atrial natriuretic factor (ANF), and alpha cardiac myosin heavy chain (MHC). Fibroblasts have readily identifiable morphology and typically express collagenase 1 , and tissue inhibitor of metalloproteinase I (TIMP-1 ). Striated muscle cells typically express contractile proteins such as skeletal α-actin, skeletal myosin heavy and light chains, and tropomyosin. Earlier myogenic markers are myoD and myogenin. Tendon and ligament tissue stains for type I collagen in a unidirectional fiber arrangement. Early tendon and chondrocyte progenitors typically express scleraxis. Adipocytes typically stain with oil red O showing lipid accumulation, and express peroxisome proliferation-activated receptor γ2 (PPARγ2), lipoprotein lipase (LPL), and fatty acid binding protein (aP2).

In other embodiments, MPC and/or MSC might be differentiated to one or more other cell types, e.g., neuronal cell types, such as neurons and glial cells, hepatocytes, pancreatic cells, e.g., beta cells, etc. Conditions for such differentiation and transdifferentiation of MPC and/or MSC have been made available in the art.

The expression of the above cell-specific markers can be detected using any suitable immunological technique known in the art, such as immuno-cytochemistry or affinity adsorption, Western blot analysis, ELISA, etc., or by any suitable biochemical assay of enzyme activity (e.g., for ALP), or by any suitable technique of measuring the quantity of the marker mRNA, e.g., Northern blot, semi-quantitative or quantitative RT-PCR, etc. Sequence data for markers listed in this disclosure are known and can be obtained from public databases such as GenBank. Calcium accumulation inside cells and deposition into matrix proteins can be measured by culturing in 45Ca²⁺, washing and re-culturing, and then determining any radioactivity present inside the cell or deposited into the extracellular matrix (U.S. Pat. No. 5,972,703), or by assaying culture substrate for mineralization using a Ca2+ assay kit (Sigma Kit #587), or as described in the examples. Wherein a cell is said to be positive for a particular marker, this means that a skilled person will conclude the presence of a distinct signal for that marker when carrying out the appropriate measurement. Where the method allows for quantitative assessment of the marker, positive cells may generate a signal that is at least 2-fold higher than such signal generated by control cells (e.g., by MSC cells before stimulating such towards further differentiation), e.g., at least 4-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold or at least 50-fold higher.

In another aspect, the invention provides primate, e.g., human, MPC and/or MSC cells, preferably mesenchymal progenitor cells capable of and/or committed to differentiation along the osteogenic lineage, and cell populations comprising such cells or substantially homologous populations of such cells, obtainable or directly obtained using the methods of the invention as described above, optionally further modified, e.g., genetically modified, as described above. Such cells or populations may be advantageously isolated, i.e., not associated with one or more cells or one or more cellular components with which the cell is associated in vivo.

In an embodiment, the said cell population comprising MPC and/or MSC, preferably mesenchymal progenitor cells capable of and/or committed to differentiation along the osteogenic lineage, cells according to the invention, comprises about 5% or more, e.g., about 10% or more of said cells, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more or about 90% or more of said cells or is a substantially homogeneous population of said cells.

In another aspect, the invention provides more differentiated cells of mesenchymal and/or connective tissue lineages, e.g., the osteocytic (bone), chondrocytic (cartilage), myocytic (muscle), tendonocytic (tendon), fibroblastic (connective tissue), adipocytic (fat) or stromogenic (marrow stroma) cell lineages, obtainable or directly obtained by further differentiation of the MSC cells as provided by the methods of the present invention. In particular, the invention provides more differentiated cells of osteogenic (bone) lineage, obtainable or directly obtained by further differentiation of the mesenchymal progenitor cells capable of and/or committed to differentiation along the osteogenic lineage provided by the methods of the present invention.

In another aspect, the invention relates to primate, e.g., human, MPC and/or MSC cells, preferably mesenchymal progenitor cells capable of and/or committed to differentiation along the osteogenic lineage, or populations comprising such cells, or substantially homogeneous populations of such cells, as obtainable or directly obtained using the methods of the invention as described above, or further derivatives obtainable or directly obtained by differentiation of the said cells, for use in therapy and/or use thereof for the manufacture of a medicament for the treatment of diseases affecting mesoderm-derived tissues.

Such diseases may include disorders affecting, e.g., bone, cartilage, muscle, tendon, fat, marrow stroma, skin or other connective tissues, preferably bone, and may represent, e.g., inborn errors, the effect of a disease condition, or the result of significant trauma. Administration of cells or differentiated derivatives thereof according to the invention can lead to tissue reconstitution or regeneration in the subject. The cells are administered in a manner that permits them to graft or migrate to the intended tissue site and reconstitute or regenerate the functionally deficient area.

By way of example and not limitation, such disorders may be any type of disease, the treatment of which may benefit from the administration of MPC and/or MSC or more differentiated derivatives thereof, to a subject having the disorder. In particular, such disorders may be characterised, e.g., by decreased formation or excessive resorption or degradation of the particular tissue, by decreased number, viability or function of cell types contributing to homeostasis of the said tissue, decreased mass of the particular tissue in a subject, compromised strength or elasticity of such tissue, etc. The term "subject" as used herein refers to a eukaryotic organism, in particular an animal or human organism. Animal subjects include prenatal forms of animals, such as, e.g., foetuses. Human subjects, e.g., human patients, may include foetuses, but usually not embryos.

Exemplary, non-limiting bone-related disorders which can benefit from administration of osteoblasts or osteoblast phenotype cells of the present invention may include local or systemic disorders, such as, osteoporosis, osteonecrosis, postmenopausal osteoporosis, senescence-associated osteoporosis, any type of fracture, e.g., non-union, mal-union, delayed union fractures or compression, conditions requiring bone fusion (e.g., spinal fusions and rebuilding), maxillo-facial fractures, bone reconstruction, e.g., after traumatic injury or cancer surgery, cranio-facial bone reconstruction, multi-site osteonecrosis, severe osteoporosis, osteopenia, osteogenesis imperfecta, osteolytic bone cancer, Paget's Disease, endocrinological disorders, hypophsophatemia, hypocalcemia, renal osteodystrophy, , osteomalacia, adynamic bone disease, rheumatoid arthritis, hyperparathyroidism, primary hyperparathyroidism, secondary hyperparathyroidism, periodontal disease, Gorham-Stout disease and McCune-AIbright syndrome, etc. By way of example and not limitation, cartilage- related disorders may include, e.g., cartilage degenerative diseases (degenerative joint disease), osteoarthritis, and osteochondritis, polychondritis. By way of example and not limitation, muscle-related disorders may include various types of myopathies, in particular atrophic or degenerative conditions, myositis, muscle necrosis, rhabdomyolysis, muscle weakness, mitochondrial myopathies, muscular dystrophy, spinal muscular athrophy, neuromuscular conditions, etc. By way of example and not limitation, tendon-related disorders may include tendonitis and tendosynovitis, or other degenerative, atrophic or autoimmune conditions affecting tendon tissue. By way of example and not limitation, skin-related conditions may include, e.g., skin burns from heat or cold, wounds, neoplastic growth, etc. Further uses of the cells of the present invention may be in further tissue engineering applications, e.g., as detailed elsewhere in this specification.

In an aspect, primate, e.g., human, MPC and/or MSC cells, preferably mesenchymal progenitor cells capable of and/or committed to differentiation along the osteogenic lineage, or populations comprising such cells, or substantially homogeneous populations of such cells, as obtainable or directly obtained using the methods of the invention as described above, or further derivatives obtainable or directly obtained by differentiation of the said cells, may be administered at a site of a tissue lesion, e.g., lesion of a connective tissue. In another aspect, the invention provides a method for preventing and/or treating a disease, comprising administration of primate, e.g., human, MPC and/or MSC cells, preferably mesenchymal progenitor cells capable of and/or committed to differentiation along the osteogenic lineage, or populations comprising such cells, or substantially homogeneous populations of such cells, as obtainable or directly obtained using the methods of the invention as described above, or further derivatives obtainable or directly obtained by differentiation of the said cells, to a subject in need of such treatment. Such administration is typically in therapeutically effective amount, i.e., generally an amount which provides the desired local or systemic effect and performance.

In a further aspect, the invention relates to a pharmaceutical composition comprising primate, e.g., human, MPC and/or MSC cells, preferably mesenchymal progenitor cells capable of and/or committed to differentiation along the osteogenic lineage, or populations comprising such cells, or substantially homogeneous populations of such cells, as obtainable or directly obtained using the methods of the invention as described above, or further derivatives obtainable or directly obtained by differentiation of the said cells, which may preferably be suitable for administration at a site of tissue lesion, e.g., connective tissue lesion.

In a further aspect, the invention relates to an arrangement comprising a surgical instrument for administration of a composition at a site of tissue lesion and further comprising the pharmaceutical composition as defined above, wherein the arrangement is adapted for administration of the pharmaceutical composition at the site of tissue lesion. For example, a suitable surgical instrument may be capable of injecting a liquid composition comprising cells of the present invention at the site of bone lesion. The cells may be introduced alone or in admixture with further components useful in the repair of tissue lesions, wounds or defects.

The pharmaceutical composition may contain further components ensuring the viability of the cells therein. In particular, the cells can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration. For general principles in medicinal formulation, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000. Choice of the cellular excipient and any accompanying elements of the composition will be adapted in accordance with the device used for administration. For example, the composition may comprise a suitable buffer system to suitable pH, e.g., near neutral pH (e.g., phosphate or carbonate buffer system), and may comprise sufficient salt to ensure iso-osmotic conditions for the cells, i.e., preventing osmotic stress. For example, suitable solution for these purposes may be phosphate-buffered saline (PBS) as known in the art. Further, the composition may comprise a carrier protein, e.g., albumin, which may increase the viability of the cells. Preferably, to ensure exclusion of non-human animal material, the albumin may be of human origin (e.g., isolated from human material or produced recombinantly). Suitable concentrations of albumin are generally known.

Further suitable uses of the MPC and/or MSC cells, preferably mesenchymal progenitor cells capable of and/or committed to differentiation along the osteogenic lineage, or cell populations, or derivatives thereof, such as more differentiated cells derived therefrom, of the invention may include the following. The cells of this invention can be used to prepare a cDNA library relatively uncontaminated with cDNA preferentially expressed in cells from other lineages, generally as well known in the art. Moreover, differential subtraction may be used to find genes particularly important in e.g., developmental pathways towards particular cell lineages. MPC and/or MSC, preferably mesenchymal progenitor cells capable of and/or committed to differentiation along the osteogenic lineage, or derivatives thereof of this invention can also be used to prepare antibodies that are specific for markers of such cells. Preparation of polyclonal and monoclonal antibodies or other antibody forms is routine known in the art. MPC and/or MSC, preferably mesenchymal progenitor cells capable of and/or committed to differentiation along the osteogenic lineage, or derivatives thereof of this invention can be used to identify expression patterns of transcripts and newly synthesized proteins that are characteristic for such cells, and may assist in directing the differentiation pathway or facilitating interaction between cells. Microarray technology, as known in the art, may be employed to define such expression profiles. MPC and/or MSC, preferably mesenchymal progenitor cells capable of and/or committed to differentiation along the osteogenic lineage, or derivatives thereof can be used to screen for factors (such as solvents, small molecule drugs, peptides, oligonucleotides) or environmental conditions (such as culture environment or manipulation) that affect the characteristics of such cells and their various progeny, e.g., differentiation characteristics or other, such as to identify differentiation and/or maturation factors capable of regulating cell growth, maintenance and differentiation towards particular lineages. Other screening applications of this invention relate to the testing of pharmaceutical compounds for their effect on maintenance or repair of MPC and/or MSC, preferably mesenchymal progenitor cells capable of and/or committed to differentiation along the osteogenic lineage, or cell types derived therefrom according to the invention. In a non-limiting illustration, derived cells with osteoblast characteristics are used to screen factors for their ability to affect calcium deposition. Screening may be done either because the compound is designed to have a pharmacological effect on the cells, or because a compound designed to have effects elsewhere may have unintended side effects on cells of this tissue type. The screening can be conducted using any of the precursor cells or terminally differentiated cells of the invention. Assessment of the activity of candidate pharmaceutical compounds generally involves combining the cells of this invention with the candidate compound, either alone or in combination with other drugs. The investigator determines any change in the morphology, marker phenotype, or functional activity of the cells that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlates the effect of the compound with the observed change. Cytotoxicity can be determined in the first instance by the effect on cell viability, survival, morphology, and the expression of certain markers and receptors. Effects of a drug on chromosomal DNA can be determined by measuring DNA synthesis or repair. Unwanted effects can also include unusual rates of sister chromatid exchange, determined by metaphase spread. Effect of cell function can be assessed using any standard assay to observe phenotype or activity MSC or differentiated derivatives thereof, such as receptor binding, matrix deposition, or production of specific proteins either in cell culture or in an appropriate animal model.

The MPC and/or MSC of the invention can also advantageously be comprised in feeder layers used for culturing pPS. In a particularly preferred embodiment, human MPC and/or MSC of the invention may be used to culture human PS, such as, e.g., hES and/or hEG cells.

The above uses may be preformed on cells derived from (presumed) genetically normal pPS cells, as well as from pPS cells containing one or more pathogenic mutations, in the latter case, such studies can elucidate pathways relevant in disorders attributable to the said mutation(s).

The following examples are provided as further non-limiting illustrations of particular embodiments of the invention.

EXAMPLES

Example 1 : Method to obtain MPC or MSC from hES cells

Three previously described hES lines were used for this study: two presumed genetically normal VUB01 (46 XY; passage 120) and VUB02 (46XY; passage 107) and one carrying a mutation for myotonic dystrophy type 1 VUB03_DM1 (46XX; passage 83) (Mateizel et al. 2006).

Undifferentiated hES cells were cultured as colonies on 0.1 % gelatine (Sigma Aldrich) coated 60mm plastic culture dishes (Becton Dickinson) containing inactivated CF1 mice embryonic fibroblasts (MEF) feeder layers (Figure 1 H). The culture medium (hES medium) consisted of 80% Knockout D-MEM (KO DMEM) (Invitrogen) supplemented with 20% Knockout-Serum Replacement (KO SR) (Invitrogen), 2 mM L-glutamine (Invitrogen), 1 % non-essential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma Aldrich) and 4 ng/ml human recombinant basic Fibroblast Growth Factor (bFGF) (Invitrogen). hES cells were grown at 37⁰C in 10%CO₂ and passaged each 4-6 days by mechanical slicing of the colonies (Mateizel et al., 2006) (Figure 1G).

For the mesenchymal differentiation, hES colonies from one culture dish were collected after collagenase type IV treatment (1 mg/ml in KO-DMEM, Invitrogen) 1 hour at 37⁰C , washed in hES medium at room temperature and then centrifuged for 5 min at 1000 rpm. The cells were resuspended in 2.5 ml differentiation medium and plated on 0.1 % gelatine coated 12-wells plastic dishes (Becton Dickinson) ("passage 1"). The differentiation medium was similar with hES culture medium except it did not contain bFGF and KO SR was substituted with FCS (20%, heat inactivated; lot 079594K; Gibco). The differentiation medium was changed every two days. After 24 days in culture (Figure 2), the cells were trypsinised (0.25% trypsin, 1 mM EDTA; Invitrogen) and plated on 0.1 % gelatine coated 60 mm plastic cultures dishes (Becton Dickinson) ("passage T). When confluent, cells were harvested with trypsin-EDTA and passaged at a 1 :2 ratio ("passage 3"). Usually, from passage 2, 3 or 4 the cells represented a morphologically homogenous population of fibroblast-like cells (Figure 3) that can be further expanded and cryopreserved.

The differentiation medium was used for the isolation and maintenance of the so obtained MPC and/or MSC. The differentiation of hES cells, isolation and maintenance of the mesenchymal progenitors was carried out at 37⁰C in 10%CO₂ (the presence of KO-DMEM may preferably require the use of 10%CO₂) typically on 0.1 % gelatine plastic culture dishes. The mesenchymal progenitors were frozen in the differentiation medium supplemented with 30% FCS and 10% DMSO (Sigma).

Example 2: Phenotypic characterisation of the MPC or MSC obtained in example 1

Immunophenotvping To detect the cell surface antigens, MPC or MSC cells from all three differentiated lines (here referred to as hMSCVUBOI , at passage 10; hMSCVUB02, at passage 9; and hMSCVUB03_DM1 , at passage 7) previously washed in PBS were incubated for 10 min at room temperature with 5 μl phycoerythrin (PE)-labelled antibody, washed with 1 % human albumin and resuspended in PBS with sodium azide: CD73 (SH3; Becton Dickinson), CD105 (SH2; Ancell), CD90 (Thy-1 ; Becton Dickinson), CD166 (ALCAM; Becton Dickinson), CD45 (LCA; Dako). For the detection of CD106 (VCAM, Becton Dickinson) and STRO-1 (R&D System) two incubation steps of 10 min each at room temperature were necessary, a first step with the unlabelled antibody and a second step with the secondary PE-labelled antibody: goat anti-mouse IgGI (Southern Biotech) and goat anti-mouse IgM (Southern Biotech), respectively. For the intracellular staining of nestin, cells were previously fixed and permeabilised with Fix&Perm kit (An Der Grub) (paraformaldehyde-based fixative, detergent- based permeabilisation) followed by 10 min incubation with 5 μl anti-human PE-labelled nestin antibody (R&D System). Approximately 10000 events were analysed for each marker by flow cytometry using a Coulter Epic XL-MCL (Analis).

Figure 4A-H illustrates that cells of the hES lines differentiated according to the invention (non-shaded histograms) are strongly positive for each CD73 (A), CD166 (D), CD105 (C), CD90 (B) which are markers characteristic of MSC, while they are negative for STRO-1 (H) CD45 (E) (marker characteristic of hematopoietic lineages) and substantially negative for CD 106 (F). Moreover, the cells are positive for nestin (G) which is a marker of neural stem cells (hMSCVUBOI , hMSCVUB02, hMSCVUB03_DM1). Shaded histograms show control detections using antibodies of the same isotype. Immuno-cvtochemistry

Cells at passage 11 (hMSCVUBOI ), passage 10 (hMSCVUB02) and passage 8 hMSCVUB03_DM1 ) were fixed with pre-chilled 100% methanol for 5 min at room temperature followed by 1 h incubation step in 3% BSA at room temperature. Staining was performed using primary antibody for vimentin (Sigma; 1 :100 dilution). Following overnight incubation, the cells were washed 3 times in PBS for 5 min each and then incubated with fluorescein- conjugated F(ab')2 fragment of Goat anti-Mouse Immunoglobulins IgG (DakoCytomation, Denmark; 1 : 100 dilution) for 2 h at room temperature in the dark.

Positive staining for vimentin, an intracellular mesenchymal marker, was found in all three above cell lines hMSCVUBOI , hMSCVUB02 and hMSCVUB03_DM1 (representative staining shown in Figure 7A).

Gene expression analysis

RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and was reverse transcribed using the First-strand cDNA Synthesis Kit (GE Healthcare, Buckinghamshire, UK) with the Λ/ofl-d(T)18 primer. A DNAse treatment (0.34 Kunitz units/μl; RNase-Free DNase Set; Qiagen) was performed for 15 min in all the samples. Quantitative RT-PCR was performed on the ABI 7500 real time PCR system (Applied Biosystems, Foster City, USA). The final reaction volume of 25 μl contained 12.5 μl of 2x TaqMan Universal Master Mix (Applied Biosystems), 1.25 μl of 2Ox Assays-on-demand Gene Expression assay mix (Applied Biosystems) and 50 ng of cDNA in 11.25 μl nuclease-free water. The primers and the probes were purchased from Applied Biosystems (Assays on demand gene expression products, Applera International Inc, Pleasanton, USA): Alpha Smooth Muscle Actin (α-SMA), Vimentin (Vim), OCT-3A (POU5F isoform 1), Nanog, and GAPDH as an endogenous control.. The following conditions were used: 2 min at 5O⁰C, 10 min at 95⁰C, 40 cycles of 15 sec at 95⁰C and 1 min at 6O⁰C. Relative quantification of gene expression between multiple samples was achieved by normalization against the endogenous control GAPDH using the ΔΔCt method. Fold changes were calculated as 2^"ΔΔCt.

Results are presented in Figure 5, showing relative amounts of Vim and α-SMA in MSC cells obtained from the three hES lines differentiated as in example 1 (compared to a primary culture of bone-marrow derived human mesenchymal stem cells (Pittenger et al. 1999) . It was herein also tested whether the above MSC or MPC cells still expressed markers of undifferentiated hES cells. Relative quantitative RT-PCR showed that MSC cells obtained from the three hES lines differentiated as in example 1 presented a significant down- regulation of the markers NANOG and POU5F-1 when compared to the corresponding undifferentiated hES cells (Figure 7B). Karyotyping

For karyotype analysis, cells at passage 16 (hMSCVUBOI ), passage 14 (hMSCVUB02) and passage 18 (hMSC_DM1 ) were cultured on 25cm² flasks (Falcon, Becton Dickinson) until 80% confluence, incubated with 3 ng/ml colcemid (Invitrogen) for 6 hours followed by 30 min treatment with hypotonic solution (KCI; 0.075M). After four fixation steps in methanol: acetic acid (3:1 ; vol/vol) cells were spread and processed for standard G-banding coloration and twenty metaphases were analysed for each line

Standard G banding karyotype analysis performed in all three differentiated populations revealed a normal 46,XY karyotype in case of hMSCVUBOI and hMSCVUB02 and 46,XX for hMSCVUB03 DM1. Example 3: Mesenchymal lineage differentiation of the cells as obtained in example 1

Osteogenic, chondrogenic and adipogenic differentiation was performed or attempted three times independently at the passages 9, 11 and 16 (hMSCVUBOI ), passages 7, 9 and 15 (hMSCVUB02) and passages 9, 11 , and 14 (hMSCVUB03_DM1). For all three differentiation protocols, a primary culture of bone-marrow derived human mesenchymal stem cells, obtained according to the protocol of Pittenger et al. 1999, at passage 6 was used as a positive control. Negative control cells were cultured in the regular growth medium and medium was replaced according to the schedule of differentiating cultures.

Osteogenic differentiation of MSC cells obtained in example 1 (Figure 6) Osteogenic differentiation was induced by plating the cells on 0.1 % gelatine coated 6 wells dishes in regular growth medium (typically 3000 cells per cm²). Cells were allowed to adhere for 4h and then medium was replaced with Osteogenic Induction Medium (Cambrex, PT3002) containing dexamethasone (0.1mM), β-glycerol phosphate (1OmM) and ascorbic acid-2- phosphate (50μM). The cells were cultured in this differentiating medium for 2-3 weeks, medium was refreshed twice a week. Negative control cells were cultured in the regular growth medium and medium was replaced according to the schedule of differentiating cultures. The differentiation potential for osteogenesis was assessed by staining intercellular calcium deposits with Von Kossa's stain. Cells were incubated with 2% AgNO₃ in the dark. After 10 min AgNO₃ was washed away and the cultures were exposed to a strong light source for 15 min thereby staining calcium deposits black. Figure 6 shows phase-contrast images of calcium accumulation in the samples treated with the induction medium (Figure 6A-C) compared with the negative control (Figure 6D-F).

Chondrogenic differentiation of MSC cells obtained in example 1

For chondrogenic differentiation 2,5 x 10⁵ MSC like cells were washed twice in Incomplete Chondrogenesis Induction Medium (Cambrex) (centrifugation 15Og for 5min), resuspended in

Complete Chondrogenesis Induction Medium and centrifuged once more at 15Og for 5 min.

Complete Chondrogenesis Induction Medium was obtained by adding TGF-β3 (0.01 μg/ml) to the incomplete medium. The resulting pellet was cultured at 37⁰C and 5% CO₂ for 2-3 weeks.

Medium was changed twice a week. To assess chondrogenic differentiation, the pellets were embedded in paraffin and processed for immunostaining for collagen type Il (Novocastra). Using the latter induction protocol, no chondrogenic differentiation (i.e., the presence of collagen type Il staining in the pellet) could be detected in these experiments for the above MPC or MSC cells obtained as in example 1. However, chondrogenic differentiation was obtained for the positive control MSC cells. Adipogenic differentiation of MSC cells obtained in example 1

Adipogenic differentiation was induced by plating the MPC or MSC cells as above on 0.1% gelatin coated 24-wells dishes at a concentration of 4x10⁴ cells per well, in the regular growth medium. When confluence was reached cells were exposed to three cycles of culture in Adipogenic Induction Medium (Cambrex) containing 1 μM dexamethasone, 0.2 mM indomethacin, 0.5 mM 3-isobutyl-1-mehyl-xanthine for 3 days followed by culture in Adipogenic Maintenance Medium (Cambrex) for 2 days. Finally, cells were maintained in culture for an additional 7 days in Adipogenic Maintenance Medium. To assess adipogenic differentiation, the cells were fixed first with 10% formalin (Merck) for 1h at room temperature, washed 3 times with PBS and incubated with 60% isopropanol (UCB) in PBS for 5 min. Finally isopropanol was removed and cells were incubated with Oil Red O (Sigma) for 5 min, after which redundant stain was washed away to end the reaction.

Using the latter induction protocol, no adipogenic differentiation (i.e., the presence of Oil Red staining for lipid droplets) could be detected in these experiments for the above MSC or MPC cells obtained as in example 1. However, adipogenic differentiation was seen for the positive control MSC cells.

Together, the consistently positive results for osteogenic differentiation, together with the absence of staining for collagen type 2 in the pellet obtained after chondrogenic differentiation and of Oil Red staining for lipid droplets after adipogenic differentiation indicates that, at least in these experiments using the MSC or MPC cells as obtained in example 1 at the specified passage numbers, such cells may represent a homogenous population of mesenchymal progenitor or stem cell like cells at an early stage of osteogenic lineage commitment.

Characterisation of osteogenic lineage commitment of MSC cells obtained as in example 1

To test potential lineage commitment of the MSC or MPC cells obtained as in example 1 , i.e., hMSCVUBOI (passage 13), hMSCVUB02 (passage 14) and hMSCVUB03_DM1 (passage 9), the cells were analysed for the expression of osteogenic markers CBFA1/RUNX2, bone sialoprotein (BSP) and osteocalcin (OCN) by relative quantitative RT-PCR. The RNA isolation, first strand cDNA synthesis and PCR conditions were the same as described in example 2. The primers and the probes were purchased from Applied Biosystems (Assays on demand gene expression products, Applera International Inc, Pleasanton, USA): CBFA1/RUNX2, osteocalcin, bone sialoprotein and GAPDH as an endogenous control. Relative quantification of gene expression between multiple samples was achieved by normalization against the endogenous control GAPDH using the ΔΔCt method. Fold changes were calculated as 2^"ΔΔCt.

CBFA1/RUNX2 is a transcription factor activated at the onset of osteogenesis and is considered to be a robust marker of osteogenic commitment (Cool et al. 2005. Stem Cells Dev 14(6): 632-42; Ducy et al. 2000. Dev Dyn 219: 461-471 ). BSP and OCN are elements of the matrix mineralization process and appear in the later stages of osteogenic differentiation, with OCN being considered a marker of mature osteoblasts.

The mRNA expression levels of CBFA1/RUNX2 show an important increase in all three cell lines when compared with the respective undifferentiated hES cells: 27.6 fold (hMSCVUBOI ), 37.2 fold (hMSCVUB02) and 132.9 fold (hMSCVUB03_DM1 ) (Figure 8A). On the other hand, expression levels of BSP and OCN were either undetectable (BSP; data not shown) or were present in this experiment at quite low levels (OCN) in all three samples as well as in their corresponding undifferentiated hES cells (Figure 8B).

To further examine the possible lineage commitment of the MSC or MPC cells obtained as in example 1 , i.e., hMSCVUBOI (passage 11 ), hMSCVUB02 (passage 10) and hMSCVUB03_DM1 (passage 8), immuno-cytochemistry was performed for collagen type I (COLL 1 ) and CBFA1/RUNX2. COLL 1 expression is regulated by CBFA1/RUNX2 and is considered to be one of the early events associated with osteoblastic differentiation (Cool et al. 2005). The cells were fixed with pre-chilled 100% methanol for 5 min at room temperature followed by 1 h incubation step in 3% BSA at room temperature. Staining was performed using primary antibody for collagen type I (Chemicon; 1 :40 dilution).

Following overnight incubation, the cells were washed 3 times in PBS for 5 min each and then incubated with Texas Red-labelled Goat anti-Rabbit (Molecular Probes, 1 :100 dilution) for 2 h at room temperature in the dark. For immunostaining of CBFA1/RUNX2, fixation and blocking steps were performed as recommended by the manufacturer (R&D Systems) followed by overnight incubation with goat anti-human CBFA1/RUNX2 (R&D Systems, 10μg/ml). The staining was performed with Alexa 488-labelled donkey anti-Goat (Molecular Probes, 1 :200 dilution) for 1 h at room temperature. Confocal scanning microscopy with an Argon-Krypton laser (488/568) (Fluoview IX70; Olympus, Belgium) was used to record the fluorescent images. All three cell lines showed positive nuclear staining for CBFA1/RUNX2 (Figure 8C) and positive cytoplasmic expression for COLL 1 (Figure 8D).

In conclusion, the results of the analysis of the above markers seems to suggest that, at least in these experiments using the MSC or MPC cells as obtained in example 1 at the specified passage numbers, such cells may represent a homogenous population of mesenchymal progenitor or stem cell like cells at an early stage of osteogenic commitment.

To further show differentiation of the mesenchymal progenitor cells of the invention towards the osteogenic lineage, cells obtained as in example 1 , i.e., VUB01-MSCL (pass. 13), VUB02- MSCL (pass. 14) and VUB03_DM1-MSCL (pass. 9), were exposed for 14 days to standard osteogenic medium and expression of bone cell specific markers was determined by quantitative RT-PCR. As seen in Figure 9, the expression of osteoblastic and osteocytic markers CBFA1 , collagen type 1 (COLLI ), alkaline phosphatase (ALP) and osteocalcin (OCN) was considerably increased in the treated cells (the y-axis value gives the relative increase of marker quantity in cells exposed to osteogenic medium vis-a-vis the respective cells not exposed to said medium). Moreover, as can be appreciated in Figure 10, increased ALP expression can be seen in VUB01-MSCL cells exposed for 14 days to osteogenic medium also by histological staining (magnification 20Ox).

Neuronal differentiation of MSC cells obtained in example 1 (Figure 11 )

Neuronal transdifferentiation of MSC cells into neuron-like cells has been previously documented (e.g., WO2004/016779). MSC of example 1 were cultured with α-MEM (Invitrogen) supplemented with 20% FBS (heat inactivated; lot 079594K; Gibco), 1 mM β- mercaptoethanol (Sigma), 5 ng/ml bFGF (Invitrogen) for 24 h and then incubated in DMEM (Invitrogen) containing 1OmM β-mercaptoethanol (Sigma), as previously described (Tsai et al. 2006. Biol Reprod 74: 545-51 ). Figure 11 illustrates that the MPC or MSC cells of the present invention induced towards neurogenesis begin to retract the cytoplasm toward the nucleus and to progressively display neuron-like extensions or processes (A), while control cells (B) do not generally display such discretely defined processes.

Claims

1. A method for differentiating primate pluripotent stem (pPS) cells into mesenchymal progenitor cells committed towards differentiation along the osteogenic pathway comprising the steps: a) plating undifferentiated pPS cells onto a substrate which allows adherence of cells thereto, b) culturing the pPS cells of a) which have adhered to the said substrate in a medium comprising serum or plasma and allowing differentiation of the pPS cells, c) passaging the cells obtained in b) at least one time and preferably two or more times, wherein the cells are replated onto a substrate which allows adherence of cells thereto and cultured in a medium comprising serum or plasma and allowing for differentiation of the said cells; wherein cells re-plated in any of the passaging steps are representative of substantially all adherent cells subjected to said passaging step.

2. The method according to claim 1 , wherein the said pPS are human embryonic stem (ES) cells or human embryonic germ (EG) cells, or established lines and sub-lines thereof.

3. The method according to any of claims 1 or 2, wherein the said substrate in one or more steps a) to c), preferably all steps a) to c), comprises one or more components of extracellular matrix.

4. The method according to any of claims 1 or 2, wherein the said substrate in one or more steps a) to c), preferably all steps a) to c), consists of or comprises gelatine.

5. The method according to any of claims 1 to 4 wherein the said serum or plasma in one or more steps b) or c), preferably both steps b) and c), is from a non-human animal, preferably a non-human mammal, even more preferably is foetal bovine serum or plasma.

6. The method according to any of claims 1 to 4 wherein the said serum or plasma in one or more steps b) or c), preferably both steps b) and c), is human serum or plasma.

7. The method according to any of claims 1 to 6, wherein the said medium in one or more steps b) or c), preferably both steps b) and c), comprises between about 15% (v/v) and about 25% (v/v), preferably about 20% (v/v), of the said serum or plasma.

8. The method according to any of claims 1 to 7, wherein the said medium in one or more steps b) or c), preferably both steps b) and c), does not contain growth factors and/or differentiation factors exogenous to, i.e., supplemental to, such factors contributed to the medium by inclusion of the said serum or plasma in the medium.

9. The method according to any of claims 1 to 8, wherein the said passaging in step c) involves detaching and dissociating of cells which recovers at least 40% or at least 50%, preferably at least 60% or at least 70%, more preferably at least 80% or at least 90% of the cells as single cells.

10. The method according to any of claims 1 to 9, further comprising differentiating of the mesenchymal progenitor cells into cells of osteocytic (bone) lineage.

11. Primate mesenchymal progenitor cells committed towards differentiation along the osteogenic pathway, preferably a cell population comprising said cells, more preferably a substantially homogeneous population of said cells, obtainable or directly obtained according to the method of any of claims 1 to 9, or cells of osteocytic lineage obtainable or directly obtained according to the method of claim 10.

12. Primate mesenchymal progenitor cells committed towards differentiation along the osteogenic pathway, preferably a cell population comprising said cells, more preferably a substantially homogeneous population of said cells, or cells of osteocytic lineage, as defined in claim 11 , for use in therapy.

13. Use of primate mesenchymal progenitor cells committed towards differentiation along the osteogenic pathway, preferably a cell population comprising said cells, more preferably a substantially homogeneous population of said cells, or cells of osteocytic lineage, as defined in claim 11 , for the manufacture of a medicament for the treatment of disorders affecting bone tissues.

14. A pharmaceutical composition comprising the primate mesenchymal progenitor cells committed towards differentiation along the osteogenic pathway, preferably a cell population comprising said cells, more preferably a substantially homogeneous population of said cells, or cells of osteocytic lineage, as defined in claim 11.