WO2005121319A1

WO2005121319A1 - Methods for production of mesodermal lineage cells

Info

Publication number: WO2005121319A1
Application number: PCT/GB2005/050089
Authority: WO
Inventors: Sakis Mantalaris; Yu-Shik Hwang; Julia Polak
Original assignee: Novathera Ltd; Imperial College Innovations Limited
Priority date: 2004-06-14
Filing date: 2005-06-14
Publication date: 2005-12-22

Abstract

The invention relates to methods of producing mesodermal lineage cells comprising incubating a pluripotent cell in the presence of HepG2-conditioned medium, an extract thereof or a factor or factors obtainable or obtained therefrom, said extract or factor(s) being capable of inducing initial differentiation of the pluripotent cell. The method can further include culture in conditions that permit formation of embryoid bodies and/or in the presence of a stimulus for mesodermal lineage differentiation. The pluripotent cell can be an embryonic stem cell (ES) or an ectoderm-like primitive (EPL) cell. Methods of the invention may be employed in screening methods. The mesodermal lineage cell can be a cardiogenic cell, mature cardiomyocyte, or a skeletal lineage cell such as an osteogenic cell, mature osteoblast, chondrogenic cell, or a mature chondrocyte.

Description

Methods for Production of Mesodermal Lineage Ceils

Technical Field

The invention relates to methods of inducing differentiation and proliferation of pluripotent cells; in particular, methods in which stem cells such as embryonic stem cells are induced to differentiate and proliferate. Using methods of the invention, initial differentiation of pluripotent cells can be controlled so that the fate of the ceils can be directed to the mesodermal lineage. Additionally, the invention provides screening methods to identify conditions, media and stimuli that influence growth and differentiation of cells. The present invention also relates to partially or terminally differentiated cells, and their uses.

Background to the Invention

The term "stem cells" describes cells that can give rise to cells of multiple tissue types. There are different types of stems cells. A single totipotent cell is formed when a sperm fertilizes an egg, this totipotent cell has the capacity to form an entire organism. In the first hours after fertilization, this cell divides into identical totipotent cells. Approximately four days after fertilization and after several cycles of cell division, these totipotent stem cells begin to specialise. When totipotent cells become more specialised, they are then termed "pluripotent". Pluripotent cells can be differentiated to every cell type in the body, but will not give rise to the placenta, or supporting tissues necessary for foetal development. Because the potential for differentiation of pluripotent cells is not "total", such cells are not termed "totipotent" and they are not embryos. Pluripotent stem cells undergo further specialization into multipotent stem cells, which are committed to differentiate to cells of a particular lineage, specialised for a particular function. Multipotent cells can be differentiated to the ceil types found in the tissue from which they were derived; for example, blood stem cells can be differentiated only into red blood cells, white blood cells and platelets.

Pluripotent stem cells (embryonic stem (ES) cells, embryonic germ (EG) cells and multipotent stem cells, such as umbilical cord stem cells and adult stem cells are powerful tools proposed for use in tissue engineering due to their ability to self-renew and their capacity for plasticity. Pluripotent stem cells, such as ES cells, can be induced to differentiate in vitro into multipotent cells of mesoderm, ectoderm and endoderm cell lineages. Mesodermal lineage cells, such as osteoblasts, chondrocytes and cardiomyocytes, are generated under the influence of osteogenic, chondrogenic, and myogenic supplements, respectively. At present, the use of pluripotent stem cells, such as ES cells, and multipotent cells in medicine is restricted by insufficient knowledge on formation of tissue-like structures and by the tendency to spontaneously differentiate towards different cell lineages; indeed this multi-lineage potential may represent a risk of heterotropic tissue formation. For clinical use, homogeneous cell populations with high purity are necessary.

For clinical therapies using pluripotent ceils to be effective, a pre-requisite is the supply of an adequate number of cells for the relevant clinical application. Undifferentiated embryonic stem cells are a promising source for key differentiated cell types, but current culture methods are either not suitable for the expansion of many undifferentiated cell populations, or do not provide an good yield of differentiated ceils.

Traditionally, embryonic stem culture protocols in 2-D cultures involve three distinct stages, first ES maintenance (i.e. self-renewal, to form stem cell colonies), then initial differentiation leading to embryoid body (EB) formation, and then further lineage-specific differentiation. Each stage requires skilled manipulation and stage-specific protocols.

For ES maintenance, originally ES cells were isolated and co-cultured on feeder layers. It was subsequently found that conditioned media can be used instead of feeder layers and that for mES cells, LIF (a trophic factor secreted from feeders) could maintain pluripotency when supplied in purified form. Assessment of ES cell pluripotency is performed by monitoring expression of the Octamer binding factor 3/4 (known as Oct-4). Oct-4 is a Pit-Oct-Unc (POU) family transcriptional regulator expressed in early embryos, germ-line cells, and undifferentiated EC (embryonic carcinoma), EG, and ES cells. Oct-4 expression in vivo is required for the development of pluripotent capacity of inner cell mass (ICM) cells and in vitro it is chemostatically controlled for the maintenance of pluripotency.

Inner cell mass (ICM) derived embryonic stem cells are differentiated into various cell types via a stage in which an embryoid body (EB) is formed. Embryoid body formation can be initiated by various stimuli, such as removal of feeder cells, removal of exposure to LIF (for murine ES cells), or removal of exposure to feeder-conditioned media. The embryoid body (EB) suspension method developed for embryonal carcinoma (EC) cells leads to formation of multi-differentiated structures, similar to post-implantation embryonic tissue, by formation of all three germ layers: mesoderm, ectoderm and endoderm. Within two to four days in suspension culture, ectoderm forms on the surface of the ICM, giving rise to structures termed "simple EBs." At around day four of differentiation, a columnar epithelium with a basal lamina develops and a central cavity forms. These structures are termed "cystic EBs" and upon continued in vitro culture, endodermal and mesodermal cells appear.

Mesodermal cells are multipotent and can be differentiated to haemopoietic and skeletal lineages, the latter including cardiomyogenic, chondrogenic and osteogenic cells. In the mesoderm, cardiogenic differentiation is known to be the first and predominant differentiation process. It is thought that cardiogenic differentiation may deter and retard other differentiation processes, such as chondrogenic and osteogenic differentiation.

Osteogenic differentiation, the in vitro formation of mineralised nodules that exhibit the morphological, ultrastructural and biochemical characteristics of woven bone formed in vivo, has been achieved by differentiation of functional osteoblasts in 2-D culture. However, 2-D culture performed in flasks and well- plates permits only a small number of cells to differentiate to the extent of being capable of organising their extracellular matrix into a structure that resembles that of bone. Furthermore, 2-D culture is fragmented, labour intensive, and requires the "judgement" of the operator during the various culture steps involved.

Static cultures, such as the 2~D methods traditionally used for ES maintenance culture and differentiation, suffer from several limitations, such as the lack of mixing, poor control options and the need for frequent feeding. Experiments in which cells are cultured in 2-D, in which normal 3-D relationships with the extracellular matrix and other cells are distorted, may result in atypical cell behaviour. Stirred suspension culture systems offer attractive advantages of scalability and relative simplicity that may influence the viability and turnover of specific stages and types of stem ceils. However, in stirred cultures of suspended cells, cell damage may result due to agitation and shear forces caused by the stirring. Processes using bioreactors to culture cells are being developed to provide dynamic cultivation systems, with controlled culture conditions, that will enable the expansion of cells in a 3-D environment. Analysing cell interactions in more natural 3-D settings promises to provide conditions closer to those in living organisms. The use of bioreactors for hESC culture has been documented and provided some preliminary evidence that dynamic, 3-D conditions may provide a suitable environment to culture ES cells.

In vitro culture of cells encapsulated in support materials has been used to try to mimic the in vivo cellular environment. Chang et al (4) pioneered bioencapsulation in the 1960's and Urn et al (5) eventually encapsulated xenograft islet cells for implantation into rats to correct diabetes. The use of alginate encapsulation has been mainly restricted to adult cells. Magyar et al (6) encapsulated mES cells in 1.1 % alginate microbeads and cultured in 2-D on tissue culture plates, i.e. in static cultures. This resulted in the formation of "discoid" colonies, which further differentiated within the beads to give cystic EBs and later to EBs containing spontaneously beating areas. When Magyar et al. encapsulated ESC into 1.6% alginate microbeads and cultured in 3-D, differentiation was found to be inhibited at the morula-like stage, so that no cystic EB could be formed within the beads; although when the ES cell colonies were released from the beads and cultured in 2-D, they were able to further differentiate into cystic EB with beating cardiomyocytes. The encapsulation of ES cells in alginate beads to generate EBs from mES cells has been attempted, but failed to yield sufficient chondrogenic differentiation (7). Mesenchymal stem ceils (MSCs) encapsulated in alginate beads have been cultured in 3-D by placing the cell beads in static flask cultures and overlaying with growth medium, to achieve chondrogenic differentiation yielding hyaline cartilage, although the proliferative capacity of the MSCs was found to be inhibited in alginate culture (8). Chondrogenic differentiation has been demonstrated in 3-D culture using human adipose-derived adult stem (ZiADAS) cells seeded in alginate or agarose hydrogels, and in porous gelatin scaffolds (Surgifoa ) (9).

Large scale production of differentiated cells from pluripotent cells requires the integration of the various culture steps. Current methods to form differentiated cells and tissues from pluripotent cells, such as ES cells, are fragmented, labour intensive and require a high level of training, which inevitably introduces operator to operator variability. At present, such methods are generally performed in 2-D cultures, which do not simulate the 3-D environment that exists in vivo. This is unsatisfactory for clinical applications as current methods of maintenance culture and of differentiation cannot produce clinically relevant cell numbers.

Therefore, there exists a need for improved methods for integrated expansion and differentiation of pluripotent cells. Such methods are necessary for efficient maintenance growth and differentiation of undifferentiated pluripotent cells and for further differentiation of partially differentiated multipotent cells, e.g. to form mesoderm lineage cells such as cardiomyocytes, osteoblasts and chondrocytes cells. For clinical bone tissue engineering applications, there is a need for methods to achieve formation of "bone nodules" (bone-like tissue) or other mesodermal lineage tissue types. According to certain aspects of the present invention, this can be achieved, e.g. in 3-D culture, using a single cell or a plurality of cells encapsulated in a support matrix.

The culture of a single cell, or clone, and the subsequent expansion and differentiation of the single clone is termed "clonality". Clonaliy-derived ES cells have been shown to differentiate in vivo when implanted into mice, but to date, attempts to culture single undifferentiated ES cells in vitro have proved to be unsuccessful. In these reported studies, the single cell cultures were performed in 2~D and the cells were not terminally differentiated to mature cells.

Currently, no methods are available for screening the effects of the cell culture environment on individual pluripotent or multipotent cells. There is thus a desire for methods of identifying the effect of cell culture conditions, media and test compounds (such as synthetic chemical entities or naturally derived materials e.g. conditioned media, growth factors) on individual cells. Furthermore, the ability to perform a large number of such screening experiments simultaneously would allow the mass screening of a great number of process variables (chemicals, concentrations, combinations).

Rathjen et al. (1 , 2, 3) have shown that upon exposure to HepG2-conditioned medium, termed MED II, murine ES cells (mES) formed early primitive ectoderm-like (EPL) cells and that exposure to MED II could enhance differentiation of adherent mES to form mesoderm, or aggregated mES to neuroectoderm. Calhoun et al. (10) have reported that treatment with MED II influences differentiation towards mesoderm in adherent human ESC culture. Thus the use of HepG2-conditioned media permits very high efficiency mesoderm formation compared to the conventional culture techniques.

The present inventors have now found that by controlling mesoderm development, subsequent mesoderm lineage differentiation can be enhanced. Statement of Invention

The present invention provides methods of inducing differentiation of pluripotent cells to cells of the mesodermal lineage comprising incubating the pluripotent cells in the presence of Hep G2 cells or HepG2-conditioned medium, an extract thereof, or a factor or factors obtainable or obtained from HepG2-conditioned medium, said extract or factor(s) being capable of causing formation of early primitive ectoderm - like cells (EPL), i.e. inducing initial differentiation of the pluripotent cells.

EPL cells express the pluripotent cell markers Oct4, SSEA1 and alkaline phosphatase. EPL formation from ES cells is accompanied by alterations in Fgf5, Gbx2 and Rex1 expression, a loss in chimaera forming ability, changes in factor responsiveness and modified differentiation capabilities, ail consistent with the identification of EPL cells as equivalent to the primitive ectoderm population of the 5.5 to 6.0 days post-coitum embryo.

Using methods of the invention a pluripotent cell can be differentiated into cells of mesodermal lineage, such as cardiomyocytes or cells of skeletal lineage, e.g. osteoblasts or chondrocytes.

The invention thus provides a method of producing a mesodermal lineage cell comprising incubating a pluripotent cell in the presence of HepG2-conditioned medium, an extract thereof or in the presence of a factor or factors obtainable or obtained therefrom, said HeρG2-conditioned medium, extract or factor(s) being capable of inducing initial differentiation of the pluripotent cell to an early primitive ectoderm-like cell (EPL).

A method of the invention for producing a mesodermal lineage cell is provided comprising: (a) culturing a pluripotent ceil in the presence of HepG2-conditioned medium, an extract thereof, or in the presence of a factor or factors obtainable or obtained from HepG2-conditioned medium, said extract or factor(s) being capable of inducing initial differentiation of the pluripotent cell, then, (b) culturing a cell obtained in (a) in conditions that permit and in the presence of a stimulus for mesodermal lineage differentiation.

Methods of the invention for producing mesodermal lineage cells may comprise a stage in which cells exposed to HepG2-conditioned medium are subsequently cultured in conditions that permit embryoid body formation. Preferably, in conditions that permit embryoid body formation, the cells are no longer exposed to HepG2-conditioned medium, an extract thereof, or a factor or factors obtainable or obtained from HepG2-conditioned medium.

Also provided is a method of producing a mesodermal lineage cell comprising:

(a) culturing a pluripotent cell in the presence of HepG2-conditioned medium, an extract thereof, or in the presence of a factor or factors obtainable or obtained from HepG2-conditioned medium, or obtained therefrom, said extract or factor(s) being capable of inducing initial differentiation of the pluripotent cell, then,

(b) culturing a cell obtained in (a) in conditions that permit formation of embryoid bodies, then,

(c) culturing a cell obtained from the embryoid body culture of (b) in conditions that permit and in the presence of a stimulus for mesodermal lineage differentiation.

In such methods, mesodermal lineage cell can be a cardiogenic cell, a cardiomyocyte, or a skeletal lineage cell, in particular an osteogenic cell or osteoblast or a chondrogenic cell or chondrocyte.

The invention also relates to a method of osteogenesis, comprising: (a) culturing a pluripotent cell in the presence of HepG2-conditioned medium, an extract thereof, or in the presence of a factor or factors obtainable or obtained from HepG2-conditioned medium, said extract or factor(s) being capable of inducing initial differentiation of the pluripotent cell, then, (b) culturing a cell obtained in (a) in conditions that permit and in the presence of a stimulus for osteogenic differentiation.

There is provided also a method of osteogenesis comprising:

(a) culturing a pluripotent cell in the presence of HepG2-conditioned medium, an extract thereof, or in the presence of a factor or factors obtainable or obtained from HepG2-conditioned medium, said extract or factor(s) being capable of inducing initial differentiation of the pluripotent cell, then,

(c) culturing a cell obtained from the embryoid body culture of (b) in conditions that permit and in the presence of a stimulus for osteogenic differentiation.

In step (b) of this osteogenesis method, culturing a cell obtained in (a) in conditions that permit formation of embryoid bodies is suitably performed for less than 5 days, preferably for 3 or 4 days or less, more preferably for around one or two days, most preferably for 12, 24 or 36 hours. Methods for embryoid body formation are well established in the art.

The stimulus for osteogenic differentiation can be a supplement provided to the culture medium, e.g. one or more of ascorbic acid,, β-glycero phosphate and dexamethasone.

Also provided by the invention is a method of chondrogenesis comprising:

(b) culturing a cell obtained in (a) in conditions that permit and in the presence of a stimulus for chondrogenic differentiation. There is provided also a method of chondrogenesis comprising:

(b) culturing a cell obtained in (a) in conditions suitable for formation of embryoid bodies, then,

(c) culturing a cell obtained from the embryoid body culture of (b) in conditions that permit and in the presence of a stimulus for chondrogenic differentiation.

In step (b) of this chondrogenesis method, culturing a cell obtained in (a) in conditions that permit formation of embryoid bodies is suitably performed for less than 5 days, preferably for 3 or 4 days or less, more preferably for around one or two days, most preferably for 12, 24 or 36 hours.

The stimulus for chondrogenic differentiation can be a supplement provided to the culture medium, e.g. monothioglycerol (MTG) and IGF-I; TGF β1 , BMP 2 or BMP 4.

Further provided is a method of cardiogenesis comprising:

(a) culturing a pluripotent cell in the presence of HepG2-conditioned medium, an extract thereof, or in the presence of a factor or factors obtainable or obtained from HepG2-conditioned medium, said extract or factor(s) being capable of inducing initial differentiation of the pluripotent cell, then, (b) culturing a cell obtained in (b) in conditions that permit and optionally in the presence of a stimulus for cardiomyogenic differentiation.

Also there is provided a method of cardiogenesis comprising: (a) culturing a pluripotent cell in the presence of HepG2-conditioned medium, an extract thereof, or in the presence of a factor or factors obtainable or obtained from HepG2-conditioned medium, said extract or factor(s) being capable of inducing initial differentiation of the pluripotent cell, then, (b) culturing a cell obtained in (a) in conditions that permit formation of embryoid bodies, then,

(c) optionally, culturing a cell obtained in (b) in conditions that permit and in the presence of a stimulus for cardiomyogenic differentiation.

In step (b) of this method of cardiogenesis, culturing a ceil obtained in (a) in conditions that permit formation of embryoid bodies is suitably performed for 5 days or more.

In a method of the invention, the pluripotent cell can be an ES cell, an in vitro or in vivo derived ICM/epiblast, an in vitro or in vivo derived primitive ectoderm, a teratocarcinoma cell, an EC cell, a primordial germ cell, an EG cell or a pluripotent cell derived by retro- or de-differentiation or by nuclear transfer.

In a method of the invention, suitably the pluripotent cell is cultured in the presence of HepG2 cells or HepG2-conditioned medium, an extract thereof, or in the presence of a factor or factors obtainable or obtained from HepG2- conditioned medium, said extract or factor(s) being capable of inducing initial differentiation of the pluripotent cell to form early primitive ectoderm-like cells, said culture being for a period of 3 to 7 days, preferably for a period or 4, 5, or 6 days.

The culture medium for HepG2 conditioning culture may comprise from 20% to 80% HepG2-conditioned medium, preferably from 30% to 70% HepG2- conditioned medium, more preferably from 40% to 60% HepG2-conditioned medium, suitably around 50% HepG2-conditioned medium.

The present inventors have found that HepG2-conditioned medium can be used in methods for enhanced induction of differentiation of pluripotent ceils such as ES cells and EPL cells to mesodermal cell lineages.

HepG2 is an established human hepatocarcinoma cell line with epithelial morphology. HepG2 cells are used routinely for a variety of biochemical and cell biological studies. HepG2 cells have been shown to be constitutive producers of Lif. HepG2 cells cultured under hypoxic conditions in 1 percent oxygen have been shown to release VEGF into the conditioned medium. HepG2 cells are believed to express multiple cytokine genes (Stonans I et al. "HepG2 human hepatoma cells express multiple cytokine genes." Cytokine 11 (2): 151-156 (1999)).

HepG2-conditioned media components that induce initial differentiation and proliferation of pluripotent cells stem cells can be identified using proteomic methods. Soluble factors, such as growth factors play an important role of ES cell differentiation to mesodermal cells. Competitive inhibition assay methods using specific antibodies for soluble factors can be used to assess the effect of individual factors and combinations of factors on cell differentiation and to identify the extracts, factor or factors that stimulate or inhibit differentiation. Studies in which gene expression is modulated can be performed to identify the factors that stimulate or inhibit differentiation of cells to the various lineages.

Production and characterisation of HepG2-conditioned medium is described in WO 99/53021 , the entire contents of which are incorporated herein by reference.

In certain embodiments of methods of the invention pluripotent ceils (e.g. embryonic stem cells) are stimulated with HepG2-conditioned media to initiate differentiation and induce formation of EPL cells, embryoid body formation is controlled by varying the duration of embryoid body formation culture, and then the ceils are stimulated to differentiate into a lineage of choice. In preferred embodiments of the invention, the method is used to enhance mesodermal cell differentiation of pluripotent cells, and in particular to improve the induction of differentiation of ES cells into myogenic lineages, such as cardiomyogenic lineages, or into osteogenic or chondrogenic lineages.

The results of the cell proliferation assay provided herein showed that from the early stages, EPL cells produced by HepG2-conditioned culture EPL cells proliferated at a much higher rate than cells that had not been exposed to HepG2-conditioned medium. A drawback of current cell expansion protocols is that the cell numbers produced are not sufficient for clinical use in therapy. An advantage of differentiation methods of the invention is that the early induction of rapid proliferation coupled with control of the duration of embryoid body formation permits production of clinically relevant numbers of desired mesoderm lineage ceils. This is particularly useful for expansion of human cell lines, as some have very slow growth rates.

The invention yet further provides a method of producing a differentiated cell comprising: culturing a pluripotent cell, which is an Early Primitive Ectoderm-like Cell (EPL) in conditions that permit and the presence of a stimulus for differentiation.

Also, there is provided a method of producing a differentiated cell comprising:

(a) culturing a pluripotent cell, which is an Early Primitive Ectoderm-like Cell (EPL), in conditions suitable for formation of embryoid bodies, then,

(b) culturing a cell obtained from the embryoid body culture of (a) in conditions that permit and in the presence of a stimulus for differentiation.

In a further aspect the invention provides a method of producing a mesodermal lineage cell comprising culturing a pluripotent cell, which is an Early Primitive Ectoderm-like Cell (EPL) in the presence of a stimulus for mesodermal lineage differentiation.

Additionally, there is provided a method of producing a mesodermal lineage cell comprising:

(a) culturing a pluripotent cell, which is a Early Primitive Ectoderm-like Cell (EPL), in conditions suitable for formation of embryoid bodies, then (b) culturing a cell obtained from the embryoid body culture of (a) in conditions that permit and in the presence of a stimulus for mesodermal lineage differentiation. EPL cells can be derived from pluripotent cells selected from an ES cell or cell ES ceil line, in vitro or in vivo derived ICM/epiblast, in vitro or in vivo derived primitive ectoderm, teratocarcinoma cells, EC cells, primordial germ cells, EG cells or pluripotent cells derived by dedifferentiation or by nuclear transfer.

EPL cells can be generated by culture of pluripotent cells, suitably ES cells, in the presence of HepG2-conditioned medium as described herein.

The mesodermal lineage cell into which EPL cells are differentiated can be cardiomyocyte and the stimulus a cardiomyogenic differentiation stimulus, or, the mesodermal lineage cell can be a skeletal lineage cell and the stimulus a skeletal lineage cell differentiation stimulus. For example the mesodermal lineage cell can be an osteogenic cell or terminally differentiated osteoblast and the stimulus an osteogenic differentiation stimulus, or the mesodermal lineage cell can be a chondrogenic cell or terminally differentiated chondrocyte and the stimulus a chondrogenic differentiation stimulus.

For cardiogenic differentiation, culturing in conditions suitable for formation of embryoid bodies is suitably performed for 5 days or more. For osteogenic differentiation or chondrogenic differentiation, culturing in conditions suitable for formation of embryoid bodies is suitably performed for less than 5 days, preferably for 3 or 4 days or less, more preferably for around one or two day, most preferably for 12, 24 or 36 hours.

Suitable culture conditions and media for growing cells in methods of the invention are well established. Suitable culture media, to which HepG2- conditioned medium can be added, include media as described in the materials and methods section and a wide variety of culture media known in the art. Suitable temperatures for culture are at or around 37°C. Culture conditions suitable for performing methods of the invention include standard culture conditions for the cell type used, e.g. for ES cell culture, suitable conditions would include the use of ES maintenance and/or differentiation culture media and environmental conditions such as 37°C and 5% CO₂. The culture conditions and media can be optimised for a given pluripotent cell type using standard methods.

For Human ES cells a feeder cell layer is conventionally required for cell adhesion and growth, the feeder layer usually being comprised of fibroblast cells. The need to use feeder layers imposes limitations on the applications to which human ES cell can be put. There is a need to purify the cells from the feeder layer cells. Feeder layer cells can also affect the behaviour of the cells of interest in biological assays.

In methods of the invention, treatment with HeρG2-conditioned medium enhanced ES cell adhesion, allowing cells with poor adhesion characteristics, such as human ES cells, to be cultured without the need to use feeder layers.

The choice of differentiation medium to provide conditions that permit differentiation of the pluripotent or multipotent cells will depend upon the type of cells employed, their requirements for growth and the stimulus required for differentiation. Any media that will support differentiation is suitable for use as a differentiation medium in methods of the invention and many suitable media are known in the art. In practice, differentiation media can be similar in composition to maintenance media used for growth (expansion) of cells, but the differentiation media will not contain a substance or substances included in the maintenance medium to suppress differentiation. Differentiation media may be enhanced by addition of a stimulus for differentiation, such as a growth factor, to maintenance media.

The use of cardiomyogenic supplements is not essential for cardiomyocyte differentiation, however cardiomyogenic differentiation can be enhanced using cardiomyogenic supplements, e.g. in the culture medium during and/or after EB formation.

For osteogenic differentiation, the differentiation medium can be, for example, any medium routinely used for osteogenic differentiation of ES cells in 2-D culture. The differentiation media used in conditions suitable for embryoid body formation and for subsequent osteogenic differentiation can be different. For mES cells, the stimulus for embryoid body formation can be removal of exposure to Lif, or where the maintenance phase is performed as co-culture, removal of exposure to Lif secreting cells. For hES cells, where the maintenance phase is performed as a co-culture, the stimulus for embryoid body formation can be removal of exposure to MEF cells.

For osteogenic differentiation to form bone nodules, culture is performed in conditions that permit osteogenic differentiation and in the presence of a stimulus for osteogenic differentiation typically for about 7 to 30 days, preferably about for 14 to 28 days.

Known in vitro inducers of osteogenic differentiation can be used; serum, ascorbate (ascorbic acid), or L-ascorbate-2-phosρhate (a long acting ascorbate analogue), β-glycerophosphate, and dexamethasone are each known to act as in vitro inducers of osteogenic differentiation. In current techniques, serum, ascorbate, and dexamethasone are absolute requirements for nodule formation whereas β-glycerophosphate promotes or enhances mineralisation The only morphological feature specific to osteoblasts is located outside the cell, in the form of a mineralised extracellular matrix. Bone nodule formation in vitro subdivided into three stages proliferation, ECM secretion/maturation and mineralisation.

In some embodiments of methods of the invention, culture conditions for mesodermal progenitor cell differentiation from pluripotent cells are optimised to enhance lineage differentiation to osteoblast or chondrocyte cells. In such methods the pluripotent cells (e.g. ES cells) are cultured in serum free media and time-dependent supplementation with BMP 4 or IGF-II without LIF is carried out throughout EB formation, e.g. in hanging drop format or otherwise.

Methods of the invention enable differentiation of pluripotent cells on a large scale, providing cell numbers required for pre-clinical and clinical trials. Conditions can be optimised for lineage differentiation of the pluripotent cells to the cell type of interest. Differentiation is achieved by formation of early primitive ectoderm cell (EPL cells), e.g. from ES cells, using conditioned media from HepG2 cell line culture, an extract thereof, or a factor or factors obtainable or obtained from Hep G2 conditioned medium, said extract or factor(s) being capable of inducing initial differentiation of the pluripotent cell to form EPL cells. The EPL cells formed can then, optionally, be subjected to culture in conditions that permit formation of embryoid bodies. The duration of embryoid body formation can be controlled to enhance subsequent differentiation to the desired cell type, cells from the embryoid body culture are then differentiated to the desired mesodermal lineage by culturing the cells in the presence of a specific stimulus for differentiation to the desired cell type. Accordingly, methods of the invention are particularly useful for stimulating pluripotent cells to differentiate into mesodermal cell lineages, such as cardiomyocytes as indicated by beating cell colonies in vitro, and chondrocytes or osteoblasts as indicated by the presence of a mineralised extracellular matrix in vitro.

According to an osteogenic method of the invention, bone nodule formation at day 21 was found to be the same for HepG2-conditioned medium-treated EPL cells and BM-treated ES cells which had been subject to 5 days of embryoid body formation culture. Where EB formation was carried out for less than 5 days, using Hep G2-conditioned EPL cells, bone nodule formation after osteogenic stimulation was found to be significantly enhanced. Without wishing to be bound by theory, it is believed that longer periods of EB formation result in extensive cardiomyocyte formation, so that fewer cells are available for differentiation into the other mesodermal lineages. Thus osteogenic differentiation or chondrogenic differentiation can be enhanced by omitting an EB formation culture step or using EB formation periods of less than 5 days, preferably less than 3 days, more preferably around 1 day, e.g. 12, 24 or 36 hours, to provide EB cultures with a greater proportion of cells available for osteogenic or chondrogenic differentiation. For cardiomyocyte differentiation, EB formation periods are preferably 3 days or more, preferably for 5 days or more, e.g. 7 days. The duration of EB formation is an important factor for enhancement of the subsequent differentiation of cells to the lineage of interest.

Positive selection for osteoblasts and negative selection for chondrocytes using sorting can be used to provide a homogeneous population of cells useful for clinical applications. For in vitro bone generation, sorted ES-derived osteoblasts are stimulated to differentiate to mature osteoblasts. In methods of the invention, differentiation to osteocyte cells can be achieved by stimulating the progenitor cells with established serum free osteogenic media containing osteogenic supplements such as ascorbic acid, β-giycerophosphate, and dexamethasone. For in vitro cartilage generation, sorted ES-derived chondrocytes are stimulated to differentiate to mature chondrocytes. For differentiation to chondrocyte cells, culture media can be supplemented with a chondrogenic supplement such as 50μM monothioglycerol (MTG), 50ng/ml IGF- I. Alternatively, chondrogenic supplements such as TGF-β1 , BMP 2 and BMP 4 can be used.

At one or more stages of differentiation methods of the invention, a pluripotent ceil may be provided encapsulated within a support matrix to form a support matrix structure, a cell obtained by initial differentiation of the pluripotent cell may be encapsulated within a support matrix to form a support matrix structure and/or in methods in which culture is performed in conditions that permit formation of embryoid bodies, a cell obtained from embryoid body culture may be encapsulated within a support matrix to form a support matrix structure. Cells may be provided encapsulated at all stages of a differentiation method of the invention. A plurality of cells may be provided encapsulated within each support matrix structure and in some embodiments a plurality of support matrix structures are provided. The support matrix structure is suitably in the form of a bead.

Methods of the invention can be used for co-culture of two or more different cell types, such as ES ceils and feeder cells. Accordingly the invention provides a method of cell culture, in which maintenance and/or differentiation of the encapsulated pluripotent or multipotent cell or ceils is performed in co-culture with a suitable second, different type of cells, e.g. feeder cells. For mES culture, the feeder cells can be HepG2. For hES, the feeder cells can be MEF (mouse embryonic fibroblasts). For hES the second type of cells can be HepG2 cells, thus in an alternative embodiment of the invention, instead of isolating and providing HepG2-conditioned medium, extracts thereof or a factor or factors obtainable or obtained therefrom, HepG2-conditioning of pluripotent cells may be achieved by co-culture of the pluripotent cells, e.g. mES or hES, with HepG2 cells. The second, different type of cells can be provided in the form of a layer and may be provided on a surface of the culture vessel and/or provided attached to, or associated with, a 3-D scaffold, such as poly lactic glycolic acid (PLGA), PMMA, PCL, polystyrene or polyurethane scaffold. The second, different type of cells can be provided as a cell or plurality of cells, encapsulated within a support matrix to form a support matrix structure, a single support matrix structure or plurality of support structures may be employed, comprising a cell or cells encapsulated within a support matrix. The support matrix structure is suitably provided in the form of a bead.

Co-culture can be performed in a 3-D bioreactor configured so that the feeder pluripotent or multipotent cells, preferably stem cells, and the co-culture second type of cells, such as feeder cells, are within common culture media, yet physically separated, for example in a two-chamber system such as a two- chamber perfused bioreactor system. Co-culture methods of the invention are useful for co-culture of stem cells, such as mES or hES cells, with feeder or other cells, e.g. cells that produce conditioning factors for induction of differentiation, or for expansion and maintenance of pluripotency, without cell contact. Specifically, one chamber can be used for the culture of the feeder or other cells, while the other chamber can be used for the culture of the ES cells. The two chambers can be separated, but in fluid communication, e.g. by a membrane that permits, and may control, mass transport between the two chambers. The chamber containing the ES cells can be supplied with conditioned medium from the feeder or other cell chamber at any proportion established as optimal. Suitably the ES cells are encapsulated, the feeder or other cells can be in suspension, encapsulated in suspension, or can be grown in 2-D culture. The invention provides a useful novel culture system for hES cells, which traditionally have been difficult to culture and have required co- culture with a feeder cell layer.

The invention also relates to methods of screening to assess the effect of the cell environment (culture conditions, media, test stimuli, compounds) on maintenance growth and/or differentiation.

Accordingly the invention further provides a method of identifying a compound capable of modulating cell maintenance and/or differentiation, identifying a stimulus capable of modulating cell differentiation, or assessing the effect of culture media and/or conditions on cell maintenance and/or differentiation, comprising a differentiated method according to the invention.

Such screening methods of the invention may involve, in at least one step, culturing a cell or cells in the presence of a test compound and assessing the effect of the test compound on cell maintenance and/or differentiation. Using this screening method of the invention it is possible to identify compounds that promote cell maintenance, by suppressing differentiation of the pluripotent or multipotent cells, and to identify compounds that promote differentiation. The test compound, or mixture of compounds, can be naturally produced or chemically synthesised.

A cell or cells may be cultured, in at least one step, in the presence of a test stimulus and the effect of the test stimulus on cell differentiation may be assessed. Using this method of the invention it is possible to identify stimuli, e.g. compounds and/or conditions, that suppress or promote differentiation.

Screening methods may also comprise culturing the cell or cells in the presence of a test medium and/or test conditions and assessing the effect of the test medium and/or test conditions on cell maintenance and/or differentiation. This method is useful for optimisation of culture conditions to enhance cell maintenance, suppress differentiation, or promote differentiation. In this method of assessment, optionally the cell can be incubated in the presence of a test compound and/or stimulus and the effect of the test compound and/or stimulus on maintenance and/or differentiation of the cell can be assessed.

In screening methods, in at least one step, a plurality of cells may be provided encapsulated within each support matrix structure; alternatively, a single cell may be provided encapsulated within each support matrix structure.

Accordingly, the invention provides the use of a pluripotent or multipotent ceil encapsulated within a support matrix for assessing the effect of a test compound or stimulus on cell maintenance and/or differentiation. The invention yet further provides use of a pluripotent or multipotent cell encapsulated within a support matrix for assessing the effect of culture media and/or conditions on cell maintenance and/or differentiation.

In screening methods of the invention, the encapsulated cell can be pluripotent cell as described herein, preferably and ES cell or EPL cell; or multipotent cell of mesoderm lineage, preferably a cardiomyogenic, osteogenic or chondrogenic cell.

The steps of differentiation or screening a method of the invention can be performed in one or more format selected from a 2-D format, e.g. a multiwell plate format, a 3-D format, or a 3-D suspension culture format. Appropriate 3-D suspension culture conditions for performing cell culture methods of the invention can be achieved using a low shear, high mixing, dynamic environment, suitably in a bioreactor. In one or more of the steps in a method of the invention, a cell or cells may be provided encapsulated within a support matrix, e.g. an alginate or alginate-based support matrix. When cells are encapsulated, culture may be performed as 3-D suspension culture in a low shear, high mixing, dynamic environment enables sufficient nutrients and gases to permeate the support matrix structure employed. Suitable bioreactor systems to provide a low shear, high mixing, dynamic environment for 3-D suspension culture include the NASA HARV bioreactor (Synthecon, USA), European Space Agency bioreactor (Fokker, Netherlands), RWV Bioreactor (Synthecon, USA) or other simulated microgravity or perfused systems, such as airlift bioreactors.

In preferred screening methods of the invention, encapsulated single cells are used, e.g. in the form of a bead, where each bead contains a single cell, such as an ES, EPL, EB or multipotent mesoderm lineage cell. By culturing a bead containing a single cell individually, suitably in multiple-well plates (which may be in array format, e.g. multi-well plates, such as 96 well plates) or micro- bioreactors. It is possible to perform multiple screens contemporaneously, to evaluate and optimise culture medium and conditions, and to screen chemically synthesised compounds, various growth factors, extracellular matrix proteins etc., for the effects that they have on cell growth and differentiation.

Screening methods can be configured so that encapsulated cells are provided in an array of culture vessels, for example as a multi-well or multi-chamber array. The culture vessel may contain a plurality of encapsulated cells, this can be achieved by providing a single support matrix structure, e.g. a bead, containing a plurality of cells; or more preferably by providing a plurality of support matrix structures in each culture vessel. In this second approach, each support matrix structure, e.g. bead, can contain a single cell or a plurality of cells. In alternative screening methods, one encapsulated cell is present in each culture vessel.

The use of methods as described herein, allows the rapid culture of single cells, e.g. ES, EPL, EB - derived or multipotent mesoderm lineage cells, in a controlled environment. This enables high throughput screening of many different culture environments in parallel or of many different cell types in the same culture environment in parallel. Suitably 5 to 20 beads each containing a single cell, can be provided in a single culture vessel, e.g. a well of a multi-well plate. Each bead constitutes an individual growth environment since a single cell within a bead will not be in direct contact with the single cells encapsulated within neighbouring beads. Placing multiple beads in a single well allows time study analyses to be performed, since each bead will be exposed to identical conditions. Culturing in multi-well plates enables screening for multiple conditions, and facilitates statistical analysis of the results. The use of robotics can facilitate the automation of the process, e.g. by feeding the cultures. Encapsulation of single cells within the beads ensures that the individual cultures are not disturbed during feeding or other manipulations.

Screening methods of the invention can be performed in 2-D culture in a culture vessel or in 3-D culture in a bioreactor, such as a HARV bioreactor or other bioreactor described herein. The use of micro-bioreactors which have micro- channels enables constant, perfused feeding of the 3-D cultures, facilitating even more elaborate screening experiments and automation. Screening methods of the invention can be performed in high throughput format.

For screening uses or methods according to the invention, the effect of a test compound, test stimulus, culture medium and/or conditions on ceil maintenance and/or differentiation can be assessed by one or more method selected from the group consisting of: microscopic examination, detection of a stage-specific antigen or antigens and, detection of gene expression levels, e.g. by RT-PCR or using a DNA or RNA microarray.

Encapsulation of a cell or cells in a support matrix, e.g. to form beads, results in an environment conducive to the maintenance of the ES or EPL cells, to EB formation, and further differentiation, e.g. osteogenic, chondrogenic or cardiogenic differentiation. Methods of the invention in which encapsulation is employed permit automation, control, optimisation and intensification of the process, enabling production of clinically relevant numbers of cells required for therapeutic applications.

Methods of the invention for differentiation to mesodermal lineage cells, i.e. cardiocytes, osteoblasts or chondrocytes can be operated on an industrial process scale for the production of specific differentiated cell types. For example, bone formation can be achieved starting with pluripotent cells, e.g. ES or EPL cells, encapsulated in a support matrix structure, e.g. alginate or alginate-based beads, and performing cultures in a bioreactor. This automated, integrated process is efficient, readily controlled and gives a significant reduction in the time taken to form bone tissues compared to prior art 2-D methods and 3-D methods.

The support matrix utilised for encapsulation is permeable to allow diffusion and mass transfer of nutrients, metabolites, and growth factors. A cell or cells encapsulated within a support matrix can be provided in the form of a bead, e.g. a generally spherical bead. By "encapsulated" it is meant that the cell or cells are entirely embedded within the support matrix. The shape of the bead is not particularly relevant, provided that the dimensions, e.g. surface area to volume ratio, are such that nutrients, metabolites, cytokines etc., can readily diffuse into/out of the bead to reach the cell or cells embedded within the bead.

It is particularly preferred that the support matrix structures, e.g. beads, are constructed of a support matrix material that remains intact during the culture time, which may be for up to 30 to 40 days, as is the case in osteogenic differentiation culture methods. The cell or cells encapsulated within the support matrix can be placed into an 3-D culture vessel such as a RWV bioreactor (Synthecon, USA) or other simulated microgravity or perfused bioreactor) and incubated in maintenance and/or differentiation medium without significant damage for prolonged periods.

Preferably the support matrix material consists of or comprises a hydroge! material, e.g, a gel-forming polysaccharide, such as an agarose or alginate, (typically in the range of from about 0.5 to about 2% w/v, preferably at from about 0.8 to about 1.5% w/v, more preferably about 0.9 to 1.2% v/v). Low viscosity alginate is preferred. The matrix may consist of alginate atone or may comprise further constituents such gelatin (typically at from about 0.05 to about 1 % w/v, preferably at from about 0.08 to about 0.5% v/v). The inclusion of gelatin assists in production of a uniform bead size and helps to maintain structural integrity. This is important because alginate hydrogels lose Ca²⁺ cations after prolonged culture, which weakens the structural integrity of the beads. Inclusion of gelatin in alginate support matrix beads enables cell- mediated contraction and packing of the scaffold material.

Alginate is a water-soluble linear polysaccharide extracted from brown seaweed and is composed of alternating blocks of 1-4 linked α-L-glucuronic and β-D- mannuronic acid residues. Alginate forms gels with most di- and multivalent cations, although Ca²⁺ is most widely used. Calcium cations take part in the interchain binding between G-blocks and give rise to a 3-dimensional network in the form of a gel.

Alginate and alginate-based support matrices, suitably in the form of beads (e.g. alginate plus gelatin beads), have been found to be particularly appropriate for use in methods of the invention, as they maintain their integrity in the culture conditions employed.

The support matrices can be modified with a variety of signals (such as laminin, collagen, or growth factors) to enhance the desired cellular behaviour. Thus, the support matrix may comprise one or more material selected from the group comprising: laminin, Bioglass™, hydroxyapatite, extracellular matrix, an extracellular matrix protein, a growth factor; an extract from another cell culture, and for osteogenic differentiation, an extract from an osteoblastic culture.

Extracellular matrix (ECM) has been used in 2^»D culture to achieve osteogenic differentiation of ES cells to (Hausemann & Pauken, 2003, Differentiation of embryonic stem cells to osteoblasts on extracellular matrix, 10^lh Annual Undergraduate research Poster Symposium, Arizona State University: http://lifesciences.asu.edu/ubep2003/participants/hausmann). Numerous growth factors are known in the art that stimulate differentiation of pluripotent stem cells such as ES cells, for example, bone morphogenesis protein 4 (BMP4) which enhances mesoderm formation and also bone formation Nakayama et al. (2003) J Cell Sci 116 (10): 2015. (http://jcs.biologists.org/cgi/reprint/116/10/2015); retinoic acid which stimulates mesoderm formation, hedgehog proteins, such as sonic hedgehog which stimulates mesoderm to osteoprogenitor differentiation and the bone morphogenesis proteins BMPs 1 to 3 and 5 to 9, which stimulate bone induction.

Calcium alginate or calcium alginate-based support matrices are favoured for osteogenic culture and differentiation. Calcium ions are used as a chelating agent in formation of the beads and may provide a local source of calcium to aid osteogenic mineralization.

The use of alginate comprising gelatin as a support matrix material for encapsulation to form support matrix structures, e.g. to form beads, is particularly preferred in methods where single cells are encapsulated, to form beads with a single cell per bead, and then cultured to form colonies.

Suitably, beads containing single cells are from about 20 to 150 microns, preferably from about 40 to about 100 microns in diameter. Beads containing a plurality of cells are generally from about 2.0 to about 2.5 millimetres, preferably about 2.3 millimetres in diameter.

The invention provides methods of integrated initial and further differentiation. In the initial differentiation phase the ES or EPL cell or cells divide and mesoderm cell numbers are increased, so that colonies of cells form within the support matrix structure, the encapsulated cells are then differentiated forming EB cells, which can then be further differentiated to cells of mesodermal lineage, all within the support matrix structure.

A method or use according the invention may further comprise freezing the encapsulated cells for storage. Encapsulated cells can be frozen using standard protocols, and may be frozen in the maintenance or differentiation medium in which they were cultured. A suitable method for freezing encapsulated cells involves cryopreservation in dimethyl sulfoxide (DMSO) using a slow freezing procedure as described by Stensvaag et al. (2004) Cell Transplantation 13 (1 ): 35-44.

The cell or cells obtained by a method of the invention can be encapsulated in a biocompatible material, so that the resulting encapsulated cells (e.g. osteogenic cells) can be administered directly to a subject patient without the need to harvest cells from the encapsulation material. For this purpose, the use of alginate or alginate-based support matrices to encapsulate cells is favoured, as alginate materials are biocompatible and alginate has FDA approval. Encapsulated cells, and in particular those encapsulated in alginate or alginate- based materials, can be administered directly to a patient, e.g. by injection or endoscopy.

Methods of the invention may further comprise liberation of a cell or cells from the support matrix. The present invention therefore provides a cell or cells so obtained. In some aspect of the invention, it is preferred that the support matrix employed can be readily dissolved to release cells, without the use of trypsinisation. In instances where it is desirable to remove the support matrix to liberate cells, hydrogel matrices, for example alginate and alginate-based matrices, are favoured as they can be readily dissolved using sodium citrate and sodium chloride solutions. Where alginate or alginate based matrices are used for encapsulation, liberation of cells can be achieved by alginate dissolution. Such gentle dissolution methods may be advantageous compared to standard enzymatic methods, such as trypsinisation, which may affect the behaviour of the cells in long-term cultures.

Pluripotent cells used in methods of the invention can be from any vertebrate source, including human sources, or non-human sources such as, non-human primate, equine, canine, bovine, porcine, caprine, ovine, piscine, rodent, murine, or avian sources. The cells may be isolated by any suitable method known in the art. The invention also provides a cell, which may be encapsulated or unencapsulated, obtained by a method of the invention, preferably by a differentiation method of the invention; the cell or encapsulated cell can be multipotent, e.g. multipotent osteogenic, chondrogenic or cardiomyogenic progenitor cells, or terminally differentiated, e.g. mature osteoblasts or mature chondrocytes.

Osteogenic cells, whether encapsulated or unencapsulated, obtained by methods of the invention are useful in the treatment of bone damage, disease and bone reconstruction. The invention also provides the use of an osteogenic cell obtained using a method of the invention as a medicament for the treatment of a disease or condition selected from: osteoporosis, bone breaks, bone fractures, bone cancer, osteocarcinoma, osteogenesis imperfecta, Paget's disease, fibrous dysplasia, bone disorders associated with hearing loss, hypophosphatasia, myeloma bone disease, osteopetrosis, over-use injury to bone, sports injury to bone and periodontal (gum) disease and reconstructive surgery, e.g. in therapeutic maxifacial surgery or in cosmetic surgery.

Further provided is the use of a chondrogenic or chondrocyte cell, whether encapsulated or unencapsulated, obtained by a method of the invention as a medicament for the treatment of a disease or condition selected from: arthritis, a cartilage disease or disorder, cartilage repair, and cosmetic reconstructive surgery. Cartilage diseases include rheumatoid arthritis and osteoarthritis, especially in articular cartilage; disorders include congenital or hereditary defects, e.g. those requiring treatment by facial reconstruction of the nasal and septal cartilage.

Yet further provided is the use of an osteogenic cell, which may be encapsulated or unencapsulated, obtained using a method of the invention in the manufacture of a medicament for the treatment of a disease or condition requiring bone reconstruction, e.g. a disease or condition selected from: osteoporosis, bone breaks, bone fractures, bone cancer, osteocarcinoma, osteogenesis imperfecta, Paget's disease, fibrous dysplasia, bone disorders associated with hearing loss, hypophosphatasia, myeloma bone disease, osteopetrosis; over-use injury to bone, sports injury to bone and periodontal (gum) disease, and reconstructive surgery.

Additionally provided is the use of an encapsulated or unencapsulated chondrogenic cell obtained by a method of the invention in the manufacture of a medicament for the treatment of a disease or disorder selected from: arthritis, a cartilage disease or disorder, cartilage repair, reconstructive surgery, cosmetic reconstructive surgery, rheumatoid and osteo arthritis.

In an further aspect, the invention provides a method of treatment of a subject comprising administration of an encapsulated or unencapsulated cell obtained according to a method of the invention. Osteogenic cells according to the invention can be administered to a subject to treat diseases or conditions requiring bone reconstruction, osteoporosis; bone breaks, bone fractures; bone cancer, osteocarcinoma, osteogenesis imperfecta, Paget's disease, fibrous dysplasia, bone disorders associated with hearing loss, hypophosphatasia, myeloma bone disease, osteopetrosis; over-use injury to bone, sports injury to bone and periodontal (gum) disease. Chondrogenic cells according to the invention can be administered to a subject to treat diseases or conditions selected from: arthritis, a cartilage disease or disorder, cartilage repair, rheumatoid and osteo arthritis.

The invention also provides a method of reconstructive surgery, which may be therapeutic or cosmetic surgery, comprising administration of a cell obtained by a method of the invention, preferably an encapsulated osteogenic or encapsulated chondrogenic cell.

Cells obtained using methods of the invention, whether unencapsulated or encapsulated, can be formulated to provide a pharmaceutical composition comprising the cells and a pharmaceutically acceptable carrier or diluent. It is preferred that the pharmaceutical composition be formulated for administration by injection, or by endoscopy. Also within the scope of the invention is a bone or cartilage tissue derived from a cell obtained by a method of the invention, suitably provided on or in a cell scaffold. Cells, preferably encapsulated cells, can be seeded onto, and/or impregnated into, a cell scaffold, which can then be implanted to allow the cells to grow in situ in the body. Such scaffolds are particularly useful in reconstructive surgery of bone and cartilage tissues.

The invention provides methods for control of lineage-specific differentiation by time-dependent treatment with conditioned medium, choice of an appropriate time period for EB formation and provision of specific supplements. Methods of the invention provide higher efficiencies of mesoderm induction and increase the yield of cells available for application in skeletal tissue engineering.

Methods of the invention can also be used for in vitro differentiation of single cells encapsulated within a support matrix, e.g. to provide homogeneous colonies or tissues. Thus, in some embodiments of methods of the invention, support matrix structures are such that a single pluripotent or multipotent cell encapsulated within a support matrix to form a support matrix structure.

A pluripotent cell, such as an ES or EPL cell, can be encapsulated into a support matrix, to provide a support matrix structure, such as a bead, containing a single cell. The encapsulated single cell can then be grown into cell colonies, EB structures can be formed, and the partially differentiated cells can eventually be differentiated into the desired mesoderm cell lineage. Mesodermal cells can be differentiated into cardiomyogenic, chondrogenic or osteogenic cells under the influence of cardiomyogenic, chondrogenic or osteogenic stimuli respectively. This permits investigation of differentiation of a single ES into the differentiated mature cell types, thus demonstrating the in vitro pluripotency potential of ES cells. This method also permits examination of 3-D embryoid body formation, cell division of ES cells, and enables investigation of the influences of the microenvironment on a single pluripotent cell. Alternatively, a plurality of cells can be provided encapsulated within a support matrix structure. This is useful for generation of large quantities of differentiated mesoderm lineage cells, e.g. for tissue engineering applications, for research, or for clinical use.

The invention provides integrated 3-D culture methods for initiation of ES differentiation, EB formation, and further differentiation. Using methods of the invention, osteogenic differentiation has been achieved in 3-D culture resulting in the formation of "bone nodules" (bone-like tissue) Chondrogenic differentiation to orm cartilage nodules has also been found to be performed efficiently in 3-D culture. Methods of the invention can be adapted for automation of the culture system, to provide low maintenance, high efficiency systems for generation of mesoderm lineage cells.

List of Figures

Figure 1 shows the scheme for the experiments that were performed, in which cells were driven towards osteogenic differentiation. A control group (BM), which included the steps for normal ES expansion, EB formation, and osteogenic differentiation, was included.

Figure 2 shows the results for cell adhesion, within 1 hour of seeding, over 95% of the cells had adhered to the surface of the tissue culture plate.

Figure 3 shows a result for the cell proliferation assay, which demonstrated that early in culture the HepG2-conditioned EPL cells proliferated at a far higher rate than cells which had not been exposed to the HepG2-conditioned medium.

Figure 4 shows the number of cardiomyocytes counted.

Figure 5 shows the 4 to 10 fold increase in the number of bone nodules formed compared with the standard culture protocol. From left to right each group of 4 bars represent 1 , 3, and 5 days of EPL derived EB formation (EPL-EB1 , EPL- EB3, EPL-EB5) and 5 days of ES derived EB formation (EB-5)

Figure 6 shows results for osteogenic differentiation of encapsulated ES cells treated initially with the HepG2 conditioned media followed by EB formation and finally by osteogenic differentiation. All images are from day 29 of osteogenic differentiation culture and are x10 objective unless otherwise stated, (a) Alzarin Red A, conventional EB formation, (b) von Kossa and Neutral Red, conventional EB formation, (c) Alizarin Red S, Hep G2. (d) von Kossa and Neutral Red, HepG2. (e) Alizarin Red S, Hep G2 (x4 objective).

Figure 7 shows the amount of osteogenic differentiation observed when encapsulated ES cells were initially cultured in differentiation medium for 5 days (n=4), compared with the amount observed when encapsulated ES cells were cultured in a conditioned medium (CM) consisting of 50% maintenance medium and 50% HepG2-conditioned medium for 5 days (n=2). In both instances the 5 day incubation was followed by culture in differentiation medium with continuous osteogenic supplements. The x- axis shows the time in days elapsed of the experiment, 15, 22, and 29 days from left to right, which relate respectively to days 7, 14 and 21 of osteogenic differentiation culture. The y-axis shows the adjusted absorbance per bead of Alizarin red S measured at 410nm. In each pair the left hand bar is the result for encapsulated ES cells cultured in differentiation medium, the right hand bar is the result for cells incubated in CM medium.

Figure 8. Morphological observation of the cartilage nodules stained by Aician blue. The various EB formation times of the HepG2 conditioned medium- treated mES cells were evaluated. Experimental groups represent: 1 day of EB formation (EPL-EB1 ), 3 days (EPL-EB3), 5 days (EPL-EB5). The control group was EB5. Figure 9. Morphological observation of cartilage nodules was evaluated with Alcian blue staining.

Figure 10. Quantification of relative Alcian Blue Stained area in cultures. Control group (EBδ) represents mES cells maintained for 4 days in conventional maintenance medium then allowed to form EBs for 5 days and then directed to the chondrogenic lineage for 15 days using the chondrogenic medium. The experimental groups were mES treated for 4 days with 50% HepG2 conditioned media (cmES) followed by 1-day (cmES^»EB1), 3-day (cmES-EB3), or 5-day (cmES-EB5) EB formation period. Finally, cmES-EB cultures were directed to the chondrogenic lineage for 15 days, similar to the control group, using chondrogenic medium. Data are shown as relative stained area ± SEM (n=6; * p<0.05 compared to EB 5).

Figure 11. Glycosaminoglycan(GAG) synthesis was assessed by DMMB assay. Control group (EBS) represents mES cells maintained for 4 days in conventional maintenance medium then allowed to form EBs for 5 days and then directed to the chondrogenic lineage for 15 days using the chondrogenic medium. The experimental groups were mES treated for 4 days with 50% HepG2 conditioned media (cmES) followed by 1-day (cmES-EBI ), 3-day (cmES-EB3), or 5-day (cmES-EB5) EB formation period. Finally, cmES-EB cultures were directed to the chondrogenic lineage for 15 days, similar to the control group, using chondrogenic medium Data are shown as relative stained area ± SEM (n=4; * p<0.05 compared to EB 5).

Figure 12. Chondrocyte marker expression of the HepG2 conditioned medium treated mES cells. Type I! collagen (green) and nuclear staining (DAPI; blue) at day 15 of culture at (a) 40x, (b) 100x and (c) 400X, respectively. OB-cadherin expression (green) and nuclear staining (DAPI; blue) at day 15 of culture at (d) 100x magnification, respectively.

Figure 13. Histological morphology of three dimensional chondrogenic differentiation was evaluated by Alcian blue and H&E staining. The experimental and control groups are as follows: Control group (EBδ) represents mES cells maintained for 4 days in conventional maintenance medium then allowed to form EBs for 5 days and encapsulated with 1.1 % alginate and 1 % gelatine and then directed to the chondrogenic lineage for 15 days using the chondrogenic medium. The experimental groups were mES treated for 4 days with 50% HeρG2 conditioned media (cmES) followed by 1-day (crnES-EB1 ), 3- day (cmES-EB3), or 5-day (cmES-EB5) EB formation period. Finally, cmES-EB cultures were and encapsulated with 1.1 % alginate and 1 % gelatine and then directed to the chondrogenic lineage for 15 days using the chondrogenic medium, similar to the control group.

Examples

Example 1

Primitive ectoderm-like (EPL) cells were formed in a 4-day culture using the HepG2-conditioned medium.

ES cells treated with HepG2-conditioned medium (EPL ceils) showed higher (approximately 10-foid) initial adhesion and proliferation activity, which diminished after 5 days in culture.

Embryoid body (EB) formation was performed in suspension culture using the standard basal medium (BM), e.g. as described in recent papers by Polak et al or by R. Bielby et al, Tissue Engineering, in press. Embryoid bodies were disrupted after 1 , 3, or 5 days by trypsinisation and the cells were seeded onto tissue culture plastic plates.

For osteogenic differentiation, both EPL- and EB-derived cultures were maintained in -MEM supplemented with 15% FBS, ascorbic acid, β- glycerophosphate, and dexamethasone for up to 21 days. Osteoblastic differentiation was assessed by bone nodule counts following Alizarin red staining for mineralised matrix after 11 , 14, 15, 16 and 21 days.

A 10-fold increase in the number of beating colonies (cardiomyocytes) was observed in the cultures grown using the conditioned medium as opposed to the basic medium (BM) at days 11 , 12, and 13 of culture.

The bone nodule assay revealed that ES cells treated with HepG2-conditioned medium (and subject to EB formation for 5 days) showed a 2-fold increase after 14 and 15 days in culture (compared to untreated ES cells). However, after 21 days there was no significant difference in bone nodule formation (between these two groups).

Materials and Methods

Medium for HepG2 culture:

DMEM high glucose with pyruvate 10% Batch tested FBS 1 % Penicillin/Streptomycin 3mM L-Glutamine

Basic Medium for TG2a cell culture:

DMEM high glucose without pyruvate 10% Batch tested FBS 1 % Penicillin/Streptomycin Additional 1 % L-glutamate 100μM (1X) β-mercaptoethanol LIF (1 μl/ml media)

Medium for Embryoid Body (EB) formation α-MEM

15% FBS

1 % Penicilin/Streptomysin Basic osteoblast medium α-MEM

1% penicillin/streptomycin (P/S) 15% FBS

Supplements: 1 μM dexamethasone (From 1000X stock) 50μg/ml ascorbic acid (from 100X stock) 10mM β-glycerophosphate (βGP) (from 100X stock)

HepG2 Culture

To obtain HepG2-conditioned medium, HepG2 cells (ATCC HB-8605) were cultured at a density of 5.0 x 10⁴ cells/cm² (3.75χ 10⁶ cells T75 flask). The conditioned medium was collected after 4 days, sterilized by filtration through a 0.22 μm filter and supplemented with 0.1 mM β-ME before use. This conditioned medium was stored at 4"C for 1-2 weeks or at approximately 20°C for up to 6 months without apparent loss of activity.

Early Primitive Ectoderm-like Cell (EPL) formation

Murine embryonic stem cells (mES) (E14/Tg2a, passage number 17-20) cells were cultured in the absence of feeders on tissue-culture flasks pre-treated with 0.2% gelatin/PBS for a minimum of 30 minutes in the TG2a medium in a 10% CO₂ humidified incubator. EPL cells were then formed and maintained for 4 days using a medium containing 50% HepG2-conditioned medium and 50% of basic Medium. E14Tg2a (ATCC No: CRL-1821) is a mouse embryonic stem cell line. It is a derivative of one of several embryonal stem cell (ES) lines developed by M. Hooper in 1987.

Embryonic stem cell culture

The murine ES cell line E14Tg2a was expanded in DMEM supplemented with 10% foetal bovine serum (FBS) in the presence of leukaemia inhibitory factor (LIF). Embryoid body formation (EB and EPL-EB formation)

After 4 days of either EPL cell or ES cell culture, HepG2-conditioned EPL cell colonies and untreated ES cell colonies were trypsinized, trypsinisation was terminated through the addition of serum containing medium, and the cells were recovered by gentle centrifugation for 10 seconds. The cells were resuspended in pre-warmed EB formation medium and were plated into non-tissue culture treated petri dishes. The cultures were fed every two days.

Adhesion assay For the adhesion assay, 24-well tissue culture polystyrene plates were coated with 0.1% (v/v) gelatin for 30 min. Cells were seeded at 6.0 x 10⁴ cells/well (3.0 x 10⁴ cells/cm²), and cultured for 1 , 2 and 4 hrs at 37°C and 5% CO₂. Cell adhesion was assessed by measuring levels of the endogenous mitochondria! dehydrogenase. Unattached cells were removed by thoroughly washing 3 times with PBS prior to the addition of 200ml of DMEM without phenol red and 40μl/well of mitochondria! dehydrogenase substrate (3-[4,5-dimethylthiazoi-2- yl]-2,5-diphenyl tetrazolium bromide). After 1 , 2 and 4 hrs incubation, mitochondrial dehydrogenase activity was stopped by the addition of 50μl 10% SDS. The plates were read at 490 nm using an ELISA reader. The number of adherent cells was determined from a standard curve of ES cells cultured on gelatin coated tissue culture plate.

It was found that within 1 hour of seeding, over 95% of the cells had adhered to the surface of the tissue culture plate (Figure 2). ES cells, especially human ES cells, are known to have poor adhesion properties; as the treated cells have improved adhesion properties, they are particularly suitable for growth on solid supports.

Proliferation assay For the proliferation assay, 24-well tissue culture polystyrene plates were coated with 0.1% (v/v) gelatin for 30 minutes. Cells were seeded at 6.0 x 10⁴ cells/well (3.0 x 10⁴ cells/cm²), and cultured for 1 , 2, 3, 4 and 5 days at 37°C and 5% CO_Ξ. The level of the endogenous mitochondrial dehydrogenase was measured to assess cell proliferation. Unattached cells were removed by thoroughly washing 3 times with PBS prior to the addition of 200μl of DMEM without phenol red and 40μl/we!l of mitochondrial dehydrogenase substrate (3- ^.S-dimethylthiazol^-ylj^.S-diphenyl tetrazolium bromide). After 2 hrs incubation, mitochondrial dehydrogenase activity was stopped by the addition of 50μl 10% SDS. Plates were read at 490 nm using ELISA reader. The number of proliferated cells was determined from a standard curve of ES cells cultured on gelatin coated tissue culture plates.

The cell proliferation assay clearly demonstrated that early in culture, the HepG2-conditioned EPL cells were proliferating at a far higher rate than cells which had not been exposed to the HepG2-conditioned medium (Figure 3).

Bone nodule formation and beating colony formation assay

After 1 , 3, and 5 days of EPL derived EB formation and 5 days of ES derived EB formation, the trypsinized cells were seeded on gelatin coated 6-well plates at a cell seeding density of 60,000 cells/well (30,000/cm²) in basic osteoblast media. After 14 days, 1 μM dexamethasone was added in basic osteoblast medium with 50μg/ml ascorbic acid and 10mM β-glycerophosphate (βGP). The media was changed every three days. Beating colonies were counted at 11 , 16, and 21 days using and inverted light microscope. Bone nodules were counted at 11 , 16, and 21 days following addition of the dexamethasone/ascorbic acid/GP osteogenic supplement. The cells were fixed with 10% of paraformaldehyde for 20 minutes, washed with PBS three times, and stained with Alizarin Red-S for 20 minutes, this was followed by a further wash with PBS. The stained bone nodules were counted using a fluorescence microscope.

Immunofluorescence staining

Osteogenic and cardiomyogenic differentiation were characterized using immunocytochemistry staining with OB-Cadherin antibody for osteogenic differentiation, and sarcomeric α-actinin antibody and tropomyosin antibody for cardiomyogenic differentiation. Cells were plated in 8-weii chamber slides at a ceil density of 24000 cells/well (30,000cells/cm²) in basic osteoblast media. After 14 days, 1 μM dexamethasone was added. The media was changed every three days. At day 11 , 16, and 21 , cells were fixed with 4% of paraformaldehyde for 10 minutes, and then washed with PBS three times. Following treatment with Triton X-100, the fixed cells were incubated with antibody, standard immunocytochemistry techniques were used. Incubation with 10% rabbit serum was employed to block non-specific binding. The cells were incubated with primary antibody overnight and this was followed by incubation with a fluorescent labelled secondary antibody for 1 hr. The stained cells were observed using a fluorescence microscope.

Cardiomyocyte differentiation

The results demonstrate that by varying EB formation time, cardiomyocyte differentiation in the EPL conditioned-treated cells can be controlled (Figure 4 shows the number of cardiomyocytes counted). Using the method of the invention, al! available cells can be directed to form the lineage of interest. Furthermore, in methods of the invention, cardiomyocyte differentiation is significantly amplified, so that cardiomyoctes can be obtained in large numbers useful for therapeutic applications, e.g. those in which the cells are used in treatment of heart damage.

Osteogenic Differentiation

There was a 4 to 10 fold increase in the number of bone nodules formed compared with the standard culture protocol (figure 5). It was found that by controlling the duration of the embryoid body formation step, osteogenic differentiation could be enhanced. The greatest enhancement of osteogenic differentiation, as shown by the greatest number of bone nodules formed, was for the HepG2-conditioned ES ceils (EPL cells) which had been subject to EB formation culture for 1 day. When EB formation was permitted for 3 days, this also provided enhanced bone nodule formation, compared to that detected for untreated ES and HepG2 treated ES (EPL) cells which had undergone EB formation culture for 5 days. Example 2 Osteogenic Differentiation of encapsulated mES cells

3D HepG2-conditioned Media (EPL-EBO)

HepG2-conditioned medium (CM) was created before the experiment was initiated. HepG2 (ATCC HB-8605) were seeded at 5 x 10 cel!s/cm² in Maintenance medium without the addition of 2-mercaptoethano! and LIF. The cells were cultured in 3 layered flasks for greater medium production and the medium was be collected after 4 days, filtered (0.2μm filter) and stored at -20°C or +4°C for no longer than two weeks. This CM was added 1:1 with Maintenance medium and used to culture encapsulated undifferentiated ES cells for 5 days. The CM was then changed to differentiation medium with continuous osteogenic supplements for a further 21 days culture.

M2 and M1 medium

The maintenance medium (M2) used was Dulbecco's modified Eagles medium (DMEM; Gibco, UK) supplemented with 10% foetal bovine serum (FBS; Gibco), 1% penicillin/streptomycin, 1 mM L-glutamine (Gibco), 0.1 mM β- mercaptoethanol (Sigma), and 1000 U/ml of leukaemia inhibitory factor (LIF; Chemicon, USA).

The differentiation medium (M1 ) was Alpha-Modified Eagles Medium (αMEM), 10% (v/v) fetal calf serum, 100units/mL penicillin and 100μg/mL streptomycin] and distributed evenly between two 90mm diameter bacteriological grade petri dishes (Bibby Sterilin, UK).

Cell encapsulation mES alginate bead encapsulation

Undifferentiated day 3mES cells (E14Tg2a) were trypsinised, counted, and resuspended at 1.5625 x 10⁶ cells/mL in 0.2μm sterile filtered, RT, 1.1 % (w/v) low viscosity alginic acid (Sigma, UK) and 0.1 % (v/v) porcine gelatin (Sigma, UK) (all dissolved in PBS, pH 7.4) solution. Using a Pharmacia peristaltic pump [Amersham Biosciences, UK (Model P-1 )], a flow rate of x20, a drop height of 30mm (tubing autoclaved and then sterilised with 1 M NaOH for 30 minutes and washed three times with sterile PBS) the cell-gel solution was passed through the peristaltic pump and dropped using a 25-gauge needle (Becton Dickinson, UK) into sterile, RT, CaCI₂ solution [120nmol/L calcium chloride (CaCI₂) (Sigma, UK) and 10mM N-(2-hydroxyethyl) piperazine-N-(2-ethane sulfonic acid) (HEPES) (Sigma, UK), in distilled water, pH 7.4]. The cell-gel solution gelled immediately on contact with the CaC!₂ solution, forming spherical beads (-2.3mm diameter after swelling). The beads remained in gently stirred CaCI₂ solution for 6-10 minutes at RT. The beads were washed three times in sterile, RT PBS and placed into M2 medium.

3D culture Undifferentiated mouse ES cells were encapsulated in microbeads on day 0 as described above, and cultured for 3 days in M2 medium in four, 50mL NASA, horizontal aspect ratio vessel (HARV) bioreactors with daily medium changes. Each reactor contained 600 beads and was rotated clockwise, continuously, at 17.5RPM from day 0 to day 21 and increased to 20RPM until day 29. This allowed the ES cells to form colonies (5-10 cells) after 3 days. To initiate differentiation, M2 medium was replaced with M1 medium on day 3 until day 8, with the medium replenished on day 6. On day 8 of culture, the M1 medium was removed and replaced with M1 medium supplemented with βadex to induce bone nodule formation and changed every 2 or 3 days until d29. All cultures were maintained in h37/5. Reactors one and two were the main experimental reactors and the third and fourth reactors were used for mineralisation quantification and ALPase activity.

Paraffin-embedding beads The beads were fixed with 4% paraformaldehyde (PFA) for 30 minutes at RT and then placed in PBS for 15 minutes prior to dehydration. The beads were taken through a sequential series of increasing ethanol concentrations to remove all the water. The ethanol was then completely replaced with neat xyiene to remove all traces of ethanol. The xylene was then replaced with paraffin saturated xylene at RT overnight. The beads in paraffin saturated xylene were then placed in an oven (60°C) for 20 minutes. The xylene was then completely replaced with liquid paraffin. The beads were then embedded, sectioned (4μm) and left at RT overnight to adhere to Vectabonded™ (Vector Laboratories, UK) glass slides. For immunocytochemistry, Alizarin Red S and Haematoxylin and Eosin staining, the paraffin was removed from the sections by immersion in xylene, decreasing ethanol concentrations and then tap water. For immunocytochemistry, the sections were autoclaved while immersed in a tri-sodium citrate dihydrate buffer (10mM, pH6.0) and allowed to cool and dry in order to retrieve the antigens.

Haematoxylin & Eosin staining 2D cells were cultured on glass Flaskette slides (Nalgene, UK). Slides were dipped in haematoxylin [(Harris' solution) BDH Laboratory Supplies, UK] for 30 seconds. Washed in running tap water for 2-3 minutes and examined for sufficient staining. Excess stain was removed by decolourising in 0.5-1 % HCl in 70% ethanol for a few seconds and then washed in tap water for 5 minutes. Slides were finally dipped in 1 % eosin (BDH Laboratory Supplies, UK) for 2 minutes and washed briefly in tap water to remove excess stain. The tissue was dehydrated by dipping in ethanol (70% once, 100% twice), cleared in xylene and mounted in DPX mountant (BDH Laboratory Supplies, UK).

Alizarin Red S / von Kossa staining

Paraffin sections were deparaffinised and hydrated in a descending series of ethanols. The sections were immersed in a 2% (w/v) Alizarin Red S, pH 4.2 (Sigma, UK) solution for 5 minutes at RT, dipped in acetone 20 times and then 1 :1 acetone/xylene 20 times, Cleared in xylene and mounted with DPX solution. Alternatively, the deparaffinised/hydrated sections were immersed in 5% (w/v) silver nitrate solution (BDH Laboratories, UK) illuminated with a 60W lamp for 1 hour at RT. Rinsed 3 times in distilled water and immersed in 5% (w/v) hypo [(sodium thiosulfate), BDH Laboratories, UK] for 5 minutes. Washed in tap water, rinsed once in distilled water and immersed in nuclear fast red (Vector Laboratories, UK) for 5 minutes at RT. Dehydrated, cleared in xylene and mounted in DPX.

Alizarin Red S (ARS) quantification

ARS quantification for 2D cultured cells was performed as documented in Gregory et al. (31 ). A slight adaptation was used for the 3D cultured beads in order to harvest the cells/tissue. Between 50 and 100 beads were isolated, counted and washed in PBS at RT. Fixed with 10% (v/v) formaldehyde for 30 minutes at RT and washed twice in distilled water. The beads were dissolved in depolymerisation buffer (21 ) for 20 minutes at RT on a rocking stage. The solution was centrifuged at 400g for 10 minutes and the pellet was washed with distilled water and centrifuged at 300g for 3 minutes. The pellet was then stained in an identical fashion to the 2D cultures.

Imaging

Morphological photographs were taken with an Olympus 1X70 inverted microscope with a Nikon CoolPix 950 digital camera and staining was captured using an Olympus BX60 upright microscope with a Zeiss axiocam.

Figure 6 shows results for osteogenic differentiation of encapsulated ES cells treated initially with the HepG2 conditioned media followed by EB formation and finally by osteogenic differentiation. All images are from day 29 of osteogenic differentiation culture and are x10 objective unless otherwise stated, (a) Alzarin Red A, conventional EB formation, (b) von Kossa and Neutral Red, conventional EB formation, (c) Alizarin Red S, Hep G2. (d) von Kossa and Neutral Red, HepG2. (e) Alizarin Red S, Hep G2 (x4 objective). Figure 7 shows the amount of osteogenic differentiation observed when encapsulated ES cells were initially cultured in differentiation medium for 5 days (n=4), compared with the amount observed when encapsulated ES cells were cultured in a conditioned medium (CM) consisting of 50% maintenance medium and 50% HepG2-conditioned medium for 5 days (n=2). In both instances the 5 day incubation was followed by culture in differentiation medium with continuous osteogenic supplements. The x- axis shows the time in days elapsed of the experiment, 15, 22, and 29 days from left to right, which relate respectively to days 7, 14 and 21 of osteogenic differentiation culture. The y-axis shows the adjusted absorbance per bead of Alizarin red S measured at 410nm. In each pair the left hand bar is the result for encapsulated ES cells cultured in differentiation medium, the right hand bar is the result for cells incubated in CM medium.

Culturing the encapsulated ES cells in HepG2-conditioned medium instead of differentiation medium for 5 days (as would be performed normally to form EB's in conventional 2D culture) results in a significant increase of mineralisation, per bead, after 14 days of osteogenic differentiation. Although there is no significant difference after 21 days. Effectively, the use of HepG2-conditioned medium reduces the time needed to form mineralised bone tissue by at least 7 days (maybe more). The histological images indicate that bone formation appears similar in both formats. It must be noted that HepG2 results are from one experiment, in duplicate but the conventional medium are in duplicate on two separate occasions.

Example 3 Chondrogenic differentiation of encapsulated mES cells

mES cell maintenance mES cells (E14/TG2a cell line) were cultured in the absence of feeder cells for 4 days in tissue-culture flasks pre-coated with 0.1 % gelatin in phosphate buffered saline (PBS; Sigma, UK). The culture medium was Dulbecco's modified Eagles medium (DMEM; Gibco, UK) supplemented with 10% foetal bovine serum (FBS; Gibco), 1 % penicillin/streptomycin, 1 mM L-glutamine (Gibco), 0.1 mM β- mercaptoethanol (Sigma), and 1000 U/mi of leukaemia inhibitory factor (LIF; Chemicon, USA). The cultures were maintained in a 5% CO₂ humidified incubator.

Collection of HepG2-conditioned media

HepG2 cells (ATCC HB-8605) were cultured in tissue-culture flasks in a 5% CO₂ humidified incubator using DMEM (Gibco) supplemented with 10% FBS, and 1 % streptomycin/penicillin (Gibco) at a seeding density of 5.0 10⁴ cells/cm². The culture medium was collected after 4 days of culture, filter- sterilised through a 0.22 μm filter, supplemented with 0.1 mM β- mercaptoethanol (Sigma), and stored at 4°C prior to use.

EPL cell formation mES cells were allowed to form EPL cells following a 4-day culture in tissue- culture flasks coated with 0.1 % gelatin using a 1 :1 culture medium containing the HepG2-conditioned medium and the standard mES cell maintenance medium described earlier. The cultures were maintained in a 5% CO₂ humidified incubator.

Embryoid body formation

Following EPL formation and ES cell maintenance culture, as described above, the EPL and ES cell cultures were trypsinised to release the colonies; trypsinisation was stopped through addition of serum containing medium before the dispersion of single cells became apparent. The cell colonies were very gently centrifuged for 10 seconds and re-suspended in pre-warmed alpha Minimal Essential Medium (α-MEM; Gibco) containing 15% FBS, and 1% penicillin/streptomycin (Gibco). The re-suspended cells were plated onto non- tissue culture treated petri dishes and cultured in a in a 5% CO₂ humidified incubator. The cultures were fed every 2 days by replacing with fresh medium.

Experimental Design

The experimental groups consisted of mES cultured for 4 days using the HepG2-conditioned media to form EPL cells followed by a 1-day (EPL-EB1 ), 3- day (EPL-EB3), or 5-day (EPL-EB5) EB formation period using the EB formation medium with a further 15 days of culture, as for the control group, using the chondrogenic medium described below.

Chondrogenic differentiation in 2D culture (micromass culture)

For micromass culture, EB cells were resuspended at density of 10x10⁶/ml and 20μl drops (2x10⁵ cells: 380000 cells/cm²) spotted onto 24 well plates. In addition, for RT-PCR and immunocytochemistry, cells were plated in 8 well chamber slide, 24 well plates and 6 well plates at a cell density of 380000 cells/cm². After 30 minutes, wells were flooded with 1 ml chondrogenic media α - MEM containing 15% foetal bovine serum (FBS; Gibco), 1 % penicillin/streptomycin, 0.4mM L-proline (Sigma), 1X Non essential amino acid (Gibco), 4500mg/L glucose, 50 μg/ml ascorbic acid, 1 μM dexamethasone and 50ng/ml IGF-I. The cultures were maintained in a 5% CO₂ humidified incubator and fed everyday.

Chondrogenic differentiation in 3D culture (Cell encapsulation with Alginate)

For 3D culture, a suspension of EB cells in 1.1 % alginate in PBS containing 1 % gelatin was allowed to gel in 10O M CaC!₂ at a cell density of 32000 ceils/bead. The alginate encapsulated cells were cultured in chondrogenic medium α-MEM containing 15% foetal bovine serum (FBS; Gibco), 1 % penicillin/streptomycin, 0.4mM L-proline (Sigma), 1X Non essential amino acid (Gibco), 4500mg/L glucose, 50 μg/ml ascorbic acid, 1 μM dexamethasone and 50ng/ml IGF-I. The cultures were maintained in a 5% CO₂ humidified incubator and fed every day.

Alcian blue staining of cartilage nodule

Cartilage nodule formation was evaluated at day 15 of culture by fixing cells with 4% paraformaldehyde (Sigma) for 20 minutes followed by three washes with PBS. The fixed cells were stained with 1 % (w/v in 3% Acetic acid, pH 2.5) Alcian blue 8 GX (Sigma) for 20 minutes and then washed with PBS. Stained cartilage nodules were observed using an inverted microscope (BX-60, Olympus, Japan). The area of stained cartilage nodule was calculated from randomly taken pictures using Photoshop program. The area of stained region was expressed as relative stained area of cartilage nodule to whole area of picture.

Dimethylmethylene Blue (DMMB) assay

To measure sulfated giycosaminoglycans (GAG) produced by cells, samples were digested with Proteinase K solution (50μg/ml proteinase K in 100mM K₂HPO (pH 8.0), Sigma) at 37°C overnight with shaking in CO₂ incubator. After digestion, samples were transferred to 1.5ml centrifuge tubes, and then proteinase K activity was inactivated by heating at 90 °C for 10 minutes. After centrifugation at 12,000g for 10min, supernatant was collected for use in GAG quantification and DNA content measurement. A 100μl aliquot of the proteinase K-treated sample solution was allowed to react with 1.4ml DMMB solution (Sigma) with vigorous vortexing for 30 minutes resulting in complexatton of GAG with DMMB. After centrifugation at 12,000g for 10min, precipitates of insoluble GAG-DMMB complex were dissolved with 1 ml DMMB decomplexation solution with vortexing for 10 minutes, and the OD₆₅₆ of the solution was measured using an ELISA reader (MRX II, Dynex Technology). For data analysis, glycosaminoglycan values were normalized to total values determined by Hoechst 33258 (Sigma) assay.

Immunocytochemistry

Chondrogenic and cardiogenic differentiation was confirmed by specific staining for OB-Cadherin, type II collagen and sarcomeric α-actinin, respectively. Cells were fixed with 4% paraformaldehyde (Sigma) for 10 minutes, washed three times with PBS, followed by permeabilisation with 0.2% Triton X-100 (Sigma), and blocking of non-specific binding by incubation with 10% (v/v) rabbit or mouse serum (Sigma). The primary antibodies, anti-OB-cadherin (diluted 1 :50 with BSA/Azide solution; Santa Cruz Biotechnology, UK), anti-type II collagen (diluted 1 :50 with BSA/Azide solution; Santa Cruz Biotechnology, UK) and anti sarcomeric α-actinin (diluted 1 :800 with BSA/Azide solution; Sigma) were incubated overnight at 4°C. Following a wash with PBS, the secondary FITC- conjugated goat anti-mouse IgG (Jackson Immunoresearch laboratory, USA) or FITC-conjugated rabbit anti-goat lgG (Santa Cruz Biotechnology) antibodies were incubated for 1 hr at room temperature. The cells were countered stained with DAPI (Sigma) to visualize the cell nucleus and were observed using epi- fluorescence on a BX-60 microscope (Olympus, Japan).

Histology

Alginate beads encapsulated cells at day 15 were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned with 4μm of depth were prepared. The tissue sections were stained with hematoxylin and eosin (H&E) and Alcian Blue.

RNA extraction and RT-PCR

RNA extraction and RT-PCR is performed to assess expression of the genes as listed in Table 1

Table 1

References

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2. Joy Rathjen, Julie-Anne Lake, Michael D. Bettess, Jenny M. Washington, Gavin Chapman and Peter D. Rathjen., Formation of a primitive ectoderm like cell population, EPL cells, from ES cells in response to biologically derived factors. Journal of Cell Science 112, 601-612 (1999)

3. Joy Rathjen and Peter D Rathjen., Mouse ES cells: experimental exploitation of pluripotent differentiation potential. Current Opinion in Genetics & Development 11 :587~594 (2001 )

4. Chang TM. SEMIPERMEABLE MICROCAPSULES. Science 1964; 146:524- 525.

5. Lim F, Moss RD. Microencapsulation of living cells and tissues. J Pharm Sci 1981 ; 70(4):351-354.

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7. Tanaka H, Murphy CL, Murphy C, Kimura M, Kawai S, Polak JM. Chondrogenic differentiation of murine embryonic stem cells: Effects of culture conditions and dexamethasone. J Cell Biochem 2004; 93(3):454.

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(10) Calhoun et al. Transcriptional profiling of initial differentiation events in human embryonic stem cells. Biochem and Biophys Research Communications 15 October 2004, Volume 323 (2), 453 - 464.

Claims

Claims:

1. A method of producing a mesodermal lineage cell comprising incubating a pluripotent cell in the presence of HepG2-conditioned medium, an extract thereof or a factor or factors obtainable or obtained therefrom, said extract or factor(s) being capable of inducing initial differentiation of the pluripotent cell.

2. A method according to claim 1 comprising:

(a) culturing a pluripotent cell in the presence of HepG2-conditioned medium, an extract thereof, or a factor or factors obtainable or obtained therefrom, said extract or factor(s) being capable of inducing initial differentiation of the pluripotent cell, then,

(b) culturing a cell obtained in (a) in the presence of a stimulus for mesodermal lineage differentiation.

3. A method according to claim 1 comprising:

(c) culturing a cell obtained from the embryoid body culture of (b) in the presence of a stimulus for mesodermal lineage differentiation.

4. A method according to any one of claims 1 to 3, wherein the mesodermal lineage cell is a cardiomyogenic cell or mature cardiomyocyte.

5. A method according to any one of claims 1 to 3, wherein the mesodermal lineage cell is a skeletal lineage cell.

6. A method according to claim 5, wherein the mesodermal lineage cell is an osteogenic cell, mature osteoblast, chondrogenic cell, or a mature chondrocyte.

7. A method of osteogenesis comprising:

(a) culturing a pluripotent cell in the presence of HepG2-conditioned medium, an extract thereof, or a factor or factors obtainable or obtained therefrom, said extract or factor(s) being capable of inducing initial differentiation of the pluripotent cell, then, (b) culturing a cell obtained in (a) in the presence of a stimulus for osteogenic differentiation.

8. A method of osteogenesis comprising:

(b) culturing a cell obtained in (a) in conditions that permit formation of embryoid bodies, then, (c) culturing a cell obtained from the embryoid body culture of (b) in the presence of a stimulus for osteogenic differentiation.

9. A method according to claim 8, wherein in (b) culturing is performed for less than 5 days.

10. A method according to any one of claims 7 to 9, wherein the stimulus for osteogenic differentiation is a supplement provided to the culture medium.

11. A method according to claim 10, wherein the supplement provided to the culture medium is ascorbic acid, β-glycerophosphate and dexamethasone.

12. A method of chondrogenesis comprising: (a) culturing a pluripotent cell in the presence of HepG2-conditioned medium, an extract thereof, or a factor or factors obtainable or obtained therefrom, said extract or factor(s) being capable of inducing initial differentiation of the pluripotent cell, then, (b) culturing a cell obtained in (a) in the presence of a stimulus for chondrogenic differentiation.

13. A method of chondrogenesis comprising:

(a) culturing a pluripotent cell in the presence of HepG2-conditioned medium, an extract thereof, or a factor or factors obtainable or obtained therefrom, said extract or factor(s) being capable of inducing initial differentiation of the pluripotent ceil, then,

(b) culturing a cell obtained in (a) in conditions suitable for formation of embryoid bodies, then, (c) culturing a cell obtained from the embryoid body culture of (b) in the presence of a stimulus for chondrogenic differentiation.

14. A method according to claim 13, wherein in (b) culturing is performed for less than 5 days.

15. A method according to any one of claims 1 to 14, wherein the stimulus for chondrogenic differentiation is a supplement provided to the culture medium.

16. A method according to claim 15, wherein the supplement provided to the culture medium is monothioglycerol (MTG) and IGF-I; TGF β, BMP 2 or BMP 4.

17. A method of cardiomyogenesis comprising:

(a) culturing a pluripotent cell in the presence of HepG2-conditioned medium, an extract thereof, or a factor or factors obtainable or obtained therefrom, said extract or factor(s) being capable of inducing differentiation of the pluripotent cell, then,

(b) culturing a cell obtained in (a), optionally in the presence of a stimulus for cardiomyogenic differentiation.

18. A method of cardiomyogenesis comprising:

(a) culturing a pluripotent ceil in the presence of HepG2-conditioned medium, an extract thereof, or in the presence of a factor or factors obtainable or obtained therefrom, said extract or factor(s) being capable of inducing initial differentiation of the pluripotent cell, then

(c) optionally, culturing a cell obtained in (b) in the presence of a stimulus for cardiomyogenic differentiation.

19. A method according to claim 18, wherein in (b) culturing is performed for 5 days or more.

20. A method according to any preceding claim, wherein the pluripotent cell is an ES cell, an in vitro or in vivo derived ICM/epiblast, an in vitro or in vivo derived primitive ectoderm, a teratocarcinoma cell, an EC cell, a primordial germ cell, an EG cell or a pluripotent cell derived by retro- or de-differentiation or by nuclear transfer.

21. A method according to any preceding claim, wherein the pluripotent cell is cultured in the presence of HepG2-conditioned medium, an extract thereof, or a factor or factors obtainable or obtained therefrom, said extract or factor(s) being capable of inducing initial differentiation of the pluripotent cell, said culture being for a period of 3 to 7 days.

22. A method according to any preceding claim, wherein the culture medium comprises from 20% to 80% HepG2-conditioned medium, preferably from 30 to 70% HepG2-conditioned medium, more preferably from 40% to 60% HepG2- conditioned medium, suitably around 50% HepG2-conditioned medium.

23. A method of producing a mesodermal lineage cell comprising culturing a pluripotent cell, which is an Early Primitive Ectoderm-like Cell (EPL), in the presence of a stimulus for mesodermal lineage differentiation.

24. A method of producing a mesodermal lineage cell comprising:

(b) culturing a cell obtained from the embryoid body culture of (a) in the presence of a stimulus for mesodermal lineage differentiation.

25. A method according to claim 23 or claim 24, wherein the mesodermal lineage cell is a cardiomyogenic cell or mature cardiomyocyte, embryoid body culture is performed for 5 or more days and the stimulus is a cardiomyogenic differentiation stimulus.

26. A method according to claim 23 or claim 24, wherein the mesodermal lineage cell is an osteogenic cell or mature osteoblast, embryoid body culture is performed for fewer than 5 days and the stimulus is an osteogenic differentiation stimulus.

27. A method according to claim 23 or claim 24, wherein the mesodermal lineage cell is a chondrogenic cell or mature chondrocyte, embryoid body culture is performed for fewer than 5 days and the stimulus is a chondrogenic differentiation stimulus.

28. A method according to any preceding claim, comprising providing a pluripotent cell encapsulated within a support matrix to form a support matrix structure.

29. A method according to any preceding claim, comprising providing a cell obtained by initial differentiation of the pluripotent cell encapsulated within a support matrix to form a support matrix structure.

30. A method according to any preceding claim in which culture is performed in conditions that permit formation of embryoid bodies, comprising providing a cell obtained from embryoid body culture encapsulated within a support matrix to form a support matrix structure.

31. A method according to any preceding claim, wherein a plurality of cells is provided encapsulated within each support matrix structure.

32. A method according to any preceding claim, wherein a plurality of support matrix structures are provided.

33. A method according to any preceding claim wherein the support matrix structure is in the form of a bead.

34. A method according to any preceding claim, wherein maintenance and/or differentiation of the encapsulated cell or ceils is performed in co-culture with a second, different type of ceils.

35. A method according to claim 34, wherein the second type of cells are feeder cells.

36. A method according to claim 34 or claim 35, wherein the second type of cells is provided in the form of a layer.

37. A method according to any one of claims claim 34 to 36, wherein the second type of cells is provided attached to, or in association with, a 3-D cell scaffold, such as a poly lactic glycolic acid (PLGA), PMMA, PCL, polystyrene or polyurethane scaffold.

38. A method according to any one of claims claim 34 to 37, wherein the second type of cells is provided as a cell or plurality of cells encapsulated within a support matrix to form a support matrix structure.

39. A method according to any preceding claim, wherein in a plurality of support matrix structures are provided.

40. A method according to any preceding claim wherein the support matrix structure is in the form of a bead.

41. A method of identifying a compound capable of modulating cell maintenance and/or differentiation, identifying a stimulus capable of modulating cell differentiation, or assessing the effect of culture media and/or conditions on cell maintenance and/or differentiation, comprising a method according to any preceding claim.

42. A method according to claim 41 comprising, in at least one step, culturing a cell or cells in the presence of a test compound and assessing the effect of the test compound on cell maintenance and/or differentiation.

43. A method according to claim 41 comprising, in at least one step, culturing a cell or cells in the presence of a test stimulus and assessing the effect of the test stimulus on cell differentiation.

44. A method according to claim 41 comprising, in at least one step, culturing the cell or cells in the presence of a test medium and/or test conditions and assessing the effect of the test medium and/or test conditions, on cell maintenance and/or differentiation.

45. A method according to claim 44, comprising, in at least one step, culturing in the presence of a test stimulus and/or test compound and assessing the effect of test stimulus and/or test compound on maintenance and/or differentiation of the cell.

46. A method according to any one of claims 41 to 45, wherein, in at least one step, cells are provided as a plurality of cells encapsulated within a support matrix structure.

47. A method according to any one of claims 41 to 46, wherein, in at least one step, a single cell is provided encapsulated within a support matrix structure.

48. A method according to any preceding claim, wherein at least one step is performed in a low shear high mixing dynamic environment.

49. A method according to any one of claims 45 to 47, wherein the encapsulated cells are provided in an array of culture vessels.

50. A method according to claim 49, wherein the array of culture vessels is a multi-well or multi-tube array.

51. A method according to claim 49 or claim 50, wherein a plurality of encapsulated cells is provided in each culture vessel.

52. A method according to any one of claims 49 to 51 , wherein a plurality of support matrix structures are provided in each culture vessel.

53. A method according to claim 49 or claim 50, wherein a single encapsulated cell is provided in each culture vessel.

54. A method according to any one of claims 46 to 53, wherein the support matrix structure is in the form of a bead.

55. A method according to any one of claims 41 to 54, wherein the effect on cell maintenance and/or differentiation is assessed by one or more method selected from the group consisting of: microscopic examination, detection of a stage-specific antigen or antigens and detection of gene expression.

56. A method according to any one of claims 28 to 55, wherein the support matrix consists of or comprises a hydrogel.

57. A method according to any one of claims 28 to 56, wherein the support matrix consists of or comprises alginate.

58. A method according to any one of claims 28 to 57, wherein the support matrix further comprises gelatin.

59. A method according to anyone of claims 56 to 58, wherein the support matrix further comprises one or more material selected from the group comprising: gelatin, laminin, Bioglass™, hydroxyapatite, extracellular matrix, an extracellular matrix protein, a growth factor; an extract from another cell culture, an extract from an osteobiastic culture.

60. A method according to any one of claims 28 to 59 further comprising freezing the encapsulated cells.

61. A method according to any one of claims 28 to 60, further comprising liberation of a cell or cells from the support matrix.

62. A method according to any preceding claim, wherein the pluripotent cell is a human pluripotent cell.

63. A method according to any one of claims 1 to 61 , wherein the pluripotent cell is a non-human pluripotent cell.

64. A method according to claims 63, wherein the pluripotent cell is of non- human primate, equine, canine, bovine, porcine, caprine, ovine, piscine, rodent, murine, or avian origin.

65. A method according to claim 64, wherein the pluripotent cell is a murine ceil.

66. A cell or encapsulated cell obtained by a method according to any preceding claim.

67. The use of a cell or encapsulated cell according to claim 66 as a medicament.

66. The use of a cell or encapsulated cell according to claim 66 in the manufacture of a medicament.

67. The use of a cardiomyocyte cell or encapsulated cardiomyocyte cell according to claim 66 in the manufacture of a medicament for the treatment of a condition associated with heart damage.

68. The use of an osteogenic cell or an encapsulated osteogenic cell according to claim 66 as a medicament for the treatment of a disease or condition selected from: osteoporosis, bone breaks, bone fractures, bone cancer, osteocarcinoma, osteogenesis imperfecta, Paget's disease, fibrous dysplasia, bone disorders associated with hearing loss, hypophosphatasia, myeloma bone disease, osteopetrosis, over-use injury to bone, sports injury to bone, periodontal (gum) disease, and reconstructive surgery such as therapeutic maxifacial surgery, or cosmetic surgery.

69. The use of a chondrogenic cell or an encapsulated chondrogenic cell according to claim 66 as a medicament for the treatment of a disease or condition selected from: arthritis, a cartilage disease or disorder, cartilage repair, cosmetic reconstructive surgery; rheumatoid and osteo arthritis.

70. The use of an osteogenic cell or encapsulated osteogenic cell according to claim 66 in the manufacture of a medicament for the treatment of a disease or condition requiring bone reconstruction.

71 , The use of an osteogenic cell or encapsulated osteogenic cell according to claim 66 in the manufacture of a medicament for the treatment of a disease or condition selected from: osteoporosis; bone breaks, bone fractures, bone cancer, osteocarcinoma, osteogenesis imperfecta, Paget's disease, fibrous dysplasia, bone disorders associated with hearing loss, hypophosphatasia, myeloma bone disease, osteopetrosis, over-use injury to bone, sports injury to bone, and periodontal (gum) disease.

72. The use of a chondrogenic cell or encapsulated chondrogenic cell according to claim 66 in the manufacture of a medicament for the treatment of a disease or condition selected from: arthritis, a cartilage disease or disorder, cartilage repair, reconstructive surgery; cosmetic reconstructive surgery, rheumatoid and osteo arthritis.

73. A method of treatment of a subject comprising administration of a cell or encapsulated cell according to claims 66 to a subject.

74. A method of treatment of a disease or condition requiring bone reconstruction comprising administration of an osteogenic cell or encapsulated osteogenic cell according to claim 66 to a subject.

75. A method of treatment a disease or condition selected from: osteoporosis; bone breaks, bone fractures; bone cancer, osteocarcinoma, osteogenesis imperfecta, Paget's disease, fibrous dysplasia, bone disorders associated with hearing loss, hypophosphatasia, myeloma bone disease, osteopetrosis; over-use injury to bone, sports injury to bone, and periodontal (gum) disease comprising administration of an osteogenic cell or encapsulated osteogenic cell according to claim 66.

76. A method of treatment of a disease or condition selected from: arthritis, a cartilage disease or disorder, cartilage repair, rheumatoid and osteo arthritis comprising administration of a chondrogenic cell or encapsulated chondrogenic cell according claim 66 to a subject.

77. A method of reconstructive surgery selected from therapeutic or cosmetic surgery comprising administration of a cell or encapsulated cell according to claim 66 to a subject.

78. A method of reconstructive surgery selected from therapeutic or cosmetic surgery comprising administration of a cell or encapsulated osteogenic or chondrogenic cell according to claim 66 to a subject.

79. A pharmaceutical composition comprising a cell or encapsulated cell according to 66 and a pharmaceutically acceptable carrier or diluent.

80. A pharmaceutical composition according to claim 79 formulated for administration by injection, or by endoscopy.

81. A. pre-filled administration device, such as a syringe, containing a pharmaceutical composition according to claim 79 or claim 80.

82. A bone or cartilage tissue derived from a cell or encapsulated cell according to claim 66.

83. A method of production of a bone or cartilage tissue comprising a method according to any one of claims 1 to 40.

84. A cell scaffold having seeded on or impregnated thereon a cell or encapsulated cells according to claim 66.

85. A method of production of a cell scaffold comprising a method according to any one of claims 1 to 40.