WO2004106499A1 - Method for expanding cells - Google Patents

Abstract

The present invention relates to a method for expanding renal progenitor cells and cartilage progenitor cells. It includes methods for proliferation and/or survival of these cells, the cells so produced and their use.

Description

Method for expanding cells

Technical Field

The present invention relates to a method for expanding renal progenitor cells and cartilage progenitor cells. It includes methods for proliferation and/or survival of these cells, the cells so produced and their use.

Background

Development of the permanent mammalian kidney (metanephros)

In mammals, including humans, three pairs of excretory organs form during fetal development in a chronological and craniocaudal order. The first excretory organs to form are the pronephroi, followed by the mesonephroi and finally the metanephroi. The metanephroi are the kidneys of adult mammals and are often referred to as the permanent kidneys.

Metanephric development involves a series of inductive interactions between the epithelial ureteric bud (UB) and the metanephric mesenchyme (MM), both of which are derived from intermediate mesoderm. The UB emerges from the Wolffian duct and grows into the adjacent MM. The MM cells induce growth and branching of the UB. Through repeated branching, the UB ultimately gives rise to the collecting duct system of the kidney, the renal calyces and pelvis, and the ureter. Simultaneously, the tips of the branching duct induce the surrounding MM to condense, epithelialise and differentiate into nephrons. Survival of the MM in vivo is dependent on the UB. If the UB does not reach the MM at the appropriate time the MM cells undergo rapid programmed cell death (apoptosis) and the animal will develop either small kidneys or no kidneys at all. Current understanding of the genetic and molecular regulation of kidney development has been described in a number of reviews (Clark and Bertram, 1999, 2000).

The precise cellular composition of the MM remains unclear and there is no clear understanding of the precise lineage relationships between MM cells (Al- Awqati and Oliver 2002). Are all cells derived from a common kidney stem cell, and do such stem cells remain in the MM at the time of the UB invasion? Or are several types of progenitor cells present, and some of these can give rise to nephrons, while others perhaps proliferate and then differentiate into the interstitial and vascular compartments of the developing kidney? While the precise lineage relationships between cells in the MM of the developing mammalian kidney remain unclear, it is known that the MM is the only mesenchyme within the developing mammal that can give rise to nephrons. Whilst other tissues such as brain and dorsal spinal cord can experimentally be shown to induce nephrogenesis in the MM, no other mesenchyme can be induced to undergo nephrogenesis (see Saxen 1987).

Studying the molecular regulation of ■metanephric development

Much research attention in recent decades has focused on identifying the factor/s that trigger nephrogenesis in the MM. Such factor/s are thought to be derived from UB tip cells. The principal in vitro assay employed since the mid- 1950s to try and identify factors that induce nephrogenesis has been whole metanephric organ culture (see Saxen 1987). With this model, whole rat or mouse metanephroi are cultured at an air/media interface. At the start of the organ culture, typically at embryonic day 14.5 (E14.5) in the rat and E12.5 in the mouse, the UB has undergone only a few branchings and the kidney contains no nephrons. However, after 4 or 5 days of culture, significant UB branching has occurred and perhaps as many as 50-60 nephrons can be seen at different stages of development. Growth and development is faster in the presence of serum, but significant growth also occurs in serum-free conditions. Using this assay, the roles of specific gene products in metanephric development can be studied. For example, exogenous purified growth factors can be added to media at known concentrations and at known time points, or the activity of specific growth factors can be inhibited through the addition to culture of neutralizing antibodies, antisense morpholino oligonucleotides or small interfering RNAs. At the end of the culture period, metanephric morphology can be assessed using a range of imaging modalities, and the effects on UB morphogenesis and nephron induction and development determined.

It is also possible to separate the UB from the MM in the earliest stages of rat and mouse metanephric development and to culture these two tissue subcompartments separately. Culture of isolated MM or UB in the presence of growth factors, neutralising antibodies and so forth provides a direct assay of the effects of these factors on MM and UB survival, growth and development. This contrasts to the situation in whole metanephric organ culture, where failure of the UB to branch and grow under experimental conditions may be the result of direct effects of factors on the UB, or alternatively, indirect effects on the MM. Similarly, failure of MM cell proliferation, induction and/or condensation in whole metanephric culture may be due to a direct effect of the specific factor of interest on the MM, or alternatively due to an indirect effect on the UB. It should be noted that the isolated MM dies in vitro in the absence of the UB. However, culture of isolated MM with heterologous tissues such as embryonic spinal cord, retina or salivary glands (among others) results not only in MM survival but also nephrogenesis. This indicates that these heterologous tissues are synthesising and secreting a factor or factors that promote MM survival and nephrogenesis. Two growth factors have been shown to promote the survival of isolated MM in vitro. These are fibroblast growth factor 2 (FGF2) (Perantoni et al. 1995) and bone morphogenetic protein 7 (BMP7) (Dudley et al. 1999)

The Transforming Growth Factor-β (TGF-β) superfamilv

The transforming growth factor-B (TGF-β) superfamily of growth factors includes several subfamilies including the TGF-βs themselves (three isoforms are found in mammals, TGF-β1 , TGF-β2, TGF-β3), the decapentaplegic protein (DPP) and Vg-1 related (DVR) family which includes the bone morphogenetic proteins (BMPs), activins (which includes activin and inhibins) and distant family members. Recent studies have demonstrated that many members of the TGF-β superfamily have roles in early embryonic development, in particular, axis formation.

The TGF-βs As mentioned above, three TGF-βs have been identified in mammals.

These TGF-βs share approximately 80% sequence homology and exhibit both overlapping and distinct spatial and temporal expression patterns during development.

The TGF-βs are multifunctional molecules with profound biological effects in many developmental processes including regulation of cell proliferation, differentiation, cell adhesion, skeletal development, haematopoiesis, inflammatory responses and wound healing. Despite eliciting common downstream pathways, the three TGF-β isoforms can exert distinct biological functions, due to different expression patterns and intrinsically different effects on specific cell types, which can be explained at least in part by differential binding to cell surface proteins which control the accessibility of the TGF-β isoforms to the receptor.

The different functions of the three TGF-β isoforms is best illustrated in the very different phenotypes of the three null mutant mice that have been generated. In brief, the three individual TGF-β mutants show distinct and only partially overlapping phenotypes. TGF-β1 knockout mice develop a severe wasting syndrome after birth associated with massive lymphocyte infiltration (Kulkarni and Karlsson 1993). The live born TGF-β1 knockout mice die approximately 3 weeks after birth. TGF-β2 knockout mice were generated by Sanford et al. (1997). These mice demonstrate a significantly different phenotype to TGF-β1 homozygous null mutants, in particular no lymphocyte infiltration is observed. Two-thirds of the TGF-β2 mutant mice die at or shortly after birth, while the remaining mice are born cyanotic. These mice demonstrate congenital heart defects and craniofacial defects. They also have abnormal limb rotation, spina bifida occulta, rib defects, eye and inner ear abnormalities, reduction in bone deposition and urogenital defects. Abnormalities in metanephric development include dilation of the renal pelvis and nephron tubules, epithelial degeneration and luminal protein casts in the nephron tubules, all of which indicate abnormalities in epithelial cell survival and/or proliferation. Sanford et al. (1997) reported no phenotypic overlap between TGF-β1 null mice and TGF-β2 null mice, indicating numerous non-compensated functions between the TGF-β isoforms. TGF-β3 knockout mice were generated by Kaartinen et al. (1995) who observed abnormalities in development of the lung and cleft palate. The lung abnormalities involved abnormalities in branching morphogenesis and respiratory epithelial cell differentiation. Sanford et al. (1997) reported that the only similarity in phenotype between TGF-β2 and TGF-β3 null mutant mice related to postnatal defects in the conducting airways of the lung. They considered that this might be related to reduced levels of surfactant in pulmonary alveoli. Foitzik et al. (1999) subsequently showed that TGF-β2 null mutant mice had major abnormalities in hair follicle morphogenesis, namely delayed hair follicle morphogenesis. In contrast, TGF-β1 null mutants displayed slightly advanced hair follicle formation, whilst TGF-β3 null mutants had normal hair. Liu et al. (2000) provided evidence that TGF-β2, but not β1 or β3 was critical for early rat lung branching. TGF-β superfamilv members and metanephric development

The effects of some TGF-β superfamily members on rat and/or mouse metanephric development have been analysed using metanephric organ culture. These include TGF-β1 (Rogers et al. 1993; Ritvos et al. 1995; Clark et al. 2001 ), BMP2 (Piscione et al. 1997), BMP4 (Miyazaki et al. 2000; Raatikainen-Ahokas et al. 2000), BMP7 (Piscione et al. 1997), and GDNF (Vega et al. 1996; Davies et al. 1999). For the most part, culture of metanephroi in the presence of TGF-β superfamily members has lead to alterations in overall metanephric growth, and/or changes in ureteric branching morphogenesis, with branching phenotypes including sprouting of ectopic branches, the growth of longer branches, and the promotion of asymmetric branching patterns. We have learned that the effects of TGF-β superfamily members on metanephric development in vitro are complex and unpredictable, and depend on such variables as the age of the explant, concentration of growth factor added and presence of absence of serum in the culture medium. The results from one superfamily member cannot be taken to predict the results of another.

Moreover, metanephric organ cultures were mostly used in these earlier studies, and they suffer from the limitations described above, viz whether an effect on the UB could be the result of a direct effect of a growth factor, or an indirect effect of the factor on MM, and vice versa.

TGF-β1 and kidney development

Addition of excess TGF-β 1 to rat metanephric organ culture inhibits nephrogenesis (Rogers et al. 1993). Nevertheless, ureteric branching continues as does nephrogenesis. In essence, the kidney continues to grow relatively normally but at a slower rate. Some of the ureteric branches appear longer than normal. In contrast, culture in the presence of a neutralising antibody to TGF-β1 accelerates nephrogenesis (Rogers et al. 1993). Ritvos et al. (1995) determined that TGF-β1 affected ureteric duct growth but not nephron tubulogenesis. We confirmed these findings and also demonstrated that addition of TGF-β1 to rat metanephric culture promotes hypertrophy of the renal capsule which consists of five or six layers of spindle-shaped cells (Clark et al. 2001 ).

TGF-β2 and kidney development Very little is known about the role of TGF-β2 in metanephric development. Pelton et al. (1991 ) described the immunostaining of TGF-β2 in the rat metanephros. Prominent staining was found in and around the UB and newly formed nephron structures such as S-shaped bodies, and these findings were confirmed by Plisov et al. (2001 ). Mariano et al. (1998), Ritvos et al. (1995) and Clark et al. (2001 ) described the localisation of TGF-β2 receptor components in the developing metanephros. The phenotype of the kidney in TGF-β2 knockout mice was described above.

In the most comprehensive study of TGF-β2 and metanephric development to date, Plisov et al. (2001) reported that TGF-β2 is secreted by inductive rat UB cells along with leukemia inhibitory factor (LIF), that fibroblast growth factor 2 (FGF2) in combination with TGF-β2 can induce the epithelialization of MM independently of exogenous LIF, and that TGF-β family members function synergistically with LIF and FGF2 to cause tubulogenesis in 72 hours, as in vivo. Specifically, Plisov et al. (2001 ) found that when TGF-β2 was added to isolated rat MM culture together with LIF, FGF2 and TGFα, epithelial tubules were induced within 72 hrs with TGF-β2 concentrations of 0.05-1 ng/ml, but above 10ng/ml it blocked tubulogenesis. FGF2 with TGF-β2 cooperated to accelerate tubulogenic differentiation. These findings extend the earlier findings of Perantoni et al. (1995) and Barasch et al. (1999) that these growth factors when used in combination can promote the survival and epithelialisation of isolated MM. However, when added independently to culture, only FGF2 was able to promote survival of isolated MM. In summary, these studies show that TGFβ2, only when combined with other factors induce phenotypic differentiation of MM towards formation of a kidney. By contrast, in the present study, we have found that isolated mouse MM can survive and proliferate and remain undifferentiated when grown in the presence of exogenous TGF-β2. Importantly to the findings of the current study, others have shown that TGF-β2 by itself did not support the survival of MM.

The mammalian renal system

The mammalian renal system serves primary roles both in the removal of catabolic waste products from the bloodstream and in the maintenance of fluid and electrolyte balances in the body. Renal failures are, therefore, life-threatening conditions in which the build-up of catabolites and other toxins, and/or the development of significant imbalances in electrolytes or fluids, may lead to the failure of other major organs systems and death. Renal failure is classified as "acute" or "chronic." Acute renal failure is defined as an abrupt cessation or substantial reduction of renal function. Acute renal failure may be due to intrinsic renal causes which involve a direct insult or injury to the kidneys, and which may entail permanent damage to the nephrons or other kidney structures.

The diagnosis and treatment of acute renal failure is as varied as its causes. If not treated, the electrolyte and fluid imbalances associated with acute renal failure may lead to life-threatening arrhythmia, congestive heart failure, or multiple organ system failures. Present therapies are typically directed at the underlying causes of the acute renal failure and management of complications. Due to the severity of acute renal failure, episodes rarely last longer than several weeks without mortality and are treated on an in-patient basis.

Chronic renal failure may be defined as a progressive, permanent and significant reduction of the glomerular filtration rate (GFR) due to a significant and continuing loss of nephrons. Chronic renal failure typically begins when chronic renal insufficiency (i.e., a permanent decrease in renal function of at least 50-60%) has resulted from some insult to the renal tissues which has caused a significant loss of nephron units. The initial insult may or may not have been associated with an episode of acute renal failure. Irrespective of the nature of the initial insult, chronic renal failure manifests a "final common path" of signs and symptoms as nephrons are progressively lost and GFR progressively declines. This progressive deterioration in renal function is slow, typically spanning many years or decades in human patients, but inevitable.

As chronic renal failure progresses, and GFR continues to decline to less than 10% of normal (e.g., 5-10 ml/min), the subject enters end-stage renal disease (ESRD). During this phase, the inability of the remaining nephrons to adequately remove waste products from the blood, while retaining useful products and maintaining fluid and electrolyte balance, leads to a rapid decline in which many organ systems, and particularly the cardiovascular system, may begin to fail. At this point, renal failure will rapidly progress to death unless the subject receives renal replacement therapy (i.e., chronic hemodialysis, continuous peritoneal dialysis, or kidney transplantation).

The 5-year survival rate for all chronic dialysis patients is approximately 40%, but for patients over 65, the rate drops to approximately 20%. Thus, there is a need for other forms of treatment for kidney disease, particularly in view of the demand, limitations associated with hemodialysis such as hospital beds, dialysis machines and costs, and shortage of donor organs. Other forms of treatment could include those currently being investigated, for instance, use of embryonic and adult stem cells for cell based therapy, or to form tissues for grafting.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

Description of the Invention

We analysed the effect of exogenous TGF-β2 on the morphological and molecular profiles of cultured rat and mouse metanephroi. Unlike others, we have found that addition of TGF- β2 to whole rat or mouse metanephroi being grown in culture results in a novel and unexpected phenotype involving the proliferation and/or survival of a population or populations of MM cells. Since MM cells are the only cells in the body capable of forming nephrons and possibly other cell types of the kidney, our results suggest that culture with TGF- β2 leads to the proliferation and survival of renal progenitor/stem cells.

We have also found that exogenous TGF-β2 assists the survival and proliferation of cultured isolated mouse MM (as distinct from whole metanephroi) grown in the absence of UB epithelial cells. Culture of isolated mouse MM in the presence of TGF-β2 promoted the survival and proliferation of the cells without marked phenotypic differentiation. As described above, MM cells grown without UB cells usually undergo rapid programmed cell death (apoptosis) in culture.

These cells may constitute the basis of, or contribute to, renal cell-based therapy, and may be useful in repairing severely diseased kidneys and/or generating de novo renal tissue, including nephrons, blood vessels and interstitium, as well as whole kidneys.

Culture of rat and mouse embryonic kidneys with exogenous TGF-β2 also gives rise to a population of cells that histologically resemble developing cartilage. These cells have the appearance of chondroblasts, stain positive for Alcian Blue and express a cartilage-specific isoform of collagen type II. This finding may provide the basis for the expansion and manipulation of embryonic cartilage cells and/or cartilage stem/progenitor cells. These cells may be of use in growing cartilage prostheses in vitro for eventual in vivo applications, and or cell-based therapy of cartilage defects in vivo and thereby contribute to cartilage repair.

TGF-β2, when added exogenously to cultures of whole rat or mouse embryonic metanephroi produces a novel phenotype. The ureteric branching morphogenesis and nephron induction and differentiation that normally occurs in the in vivo and cultured kidney does not occur. Rather, following a few days of culture, the kidney consists of a mass of mesenchymal cells, and in particular two populations of cells that we have called Type 1 and Type 2. Importantly, nephron induction has ceased.

In contrast, culture of rat or mouse metanephroi with other members of the TGF-β superfamily of growth factors does not yield this result. The major findings of this study are: (1) culture of whole rat or mouse metanephroi with TGF-B2 alone results in a completely novel and unexpected kidney phenotype in which ureteric branching and nephrogenesis is stopped, and the kidney is mostly comprised of morphologically undifferentiated mesenchymal cells - no other single factor gives this phenotype; (2) these undifferentiated mesenchymal cells may include a population or populations of kidney stem/progenitor cells, based on the fact that (i) these kidneys express several genes known to be expressed in normal MM (BMP4, GDNF, PAX2, Wnt4, WT1 , BF2), and (ii) the fact that it is only MM cells in the body that can give rise to kidneys. Kidney stem or progenitor cells would have kidney, nephron, renal vasculature capabilities or the like; (3) culture of whole metanephroi with TGF-B2 promotes the development of cartilage in that at least some cells in the MM can be redirected from a renal fate to a cartilage fate; (4) culture of isolated MM with TGF- B2 leads to the survival and proliferation of subpopulations of MM cells. Thus, in a first aspect the present invention provides a method for maintaining the survival of a renal progenitor population which method comprises treating the population with TGF-β2. Also provided is use of TGF-β2 in maintaining the survival of a renal progenitor population. In a second aspect the present invention provides a method for expanding a renal progenitor population which method comprises treating the population with TGF-β2. Also provided is use of TGF-β2 in expanding a renal progenitor population.

In a third aspect the present invention provides use of TGF-β2 to drive MM cells towards a renal and/or a cartilage phenotype by incubating the MM cells in the presence of TGF-β2. Incubation in the presence of TGF-β2 from culture initiation promotes formation of both renal progenitor cells and cartilage progenitor cells whereas addition of TGF-β2 at any time promotes formation of renal progenitor cells. In a fourth aspect the present invention provides a method for inducing the production of multiple mesenchymal lineages from kidney cells which method comprises treating the kidney cells with TGF-β2. In a preferred embodiment the mesenchymal lineage is a renal or cartilage lineage. Incubation in the presence of TGF-β2 from culture initiation promotes formation of renal progenitor cells and cartilage progenitor cells whereas addition of TGF-β2 at any time promotes formation of renal progenitor cells.

In a fifth aspect, the present invention provides a method for expanding and isolating renal and/or cartilage progenitor cells which method comprises culturing the cells in the presence of exogenous TGF-β2 to expand the cells and then isolating the cells by a suitable cell isolation technique. Incubation in the presence of TGF-β2 from culture initiation promotes formation of renal progenitor cells and cartilage progenitor cells whereas addition of TGF-β2 at any time promotes formation of renal progenitor cells. Expansion of these cells permits their molecular characterisation, identification of specific markers and the use of these markers in isolating separate populations of these cells. Expanded, isolated populations are particularly desirable for clinical purposes.

In a sixth aspect the present invention provides use of TGF-β2 treated mesenchymal cells as a source of renal and/or cartilage progenitor cells. The renal lineage cells may ultimately be of use in repairing diseased kidneys and/or generating de novo renal tissue, nephrons or kidneys for patients with kidney disease.

The cartilage lineage cells may be of use in growing in vitro cartilage segments or prostheses for transplantation into patients, or alternatively for cell- based therapy for patients with cartilage defects.

The present invention provides in a seventh aspect cells for use in renal cell-based therapy comprising renal mesenchymal cells or their derivatives produced or maintained by a method of the invention. Culture in medium lacking TGF-β2 followed by addition of exogenous TGF- β2 leads to the proliferation of these cells. ι

In an eighth aspect the present invention provides a method for repairing severely diseased kidneys and/or generating de novo renal tissue, nephrons or kidneys which method comprises use of renal mesenchymal cells produced or maintained by a method of the invention.

In a ninth aspect the present invention provides cartilage mesenchymal cells produced or maintained by a method of the invention for use in growing cartilage prostheses in vitro for in vivo applications, and/ or cell-based therapy of cartilage defects in vivo. Typically, these cells are generated by adding TGF-β2 from the beginning of the culture. Long term culture with TGF-β2 enhances survival and/or proliferation of these cells.

The present invention provides, in a tenth aspect, a method of cartilage repair comprising use of cartilage mesenchymal cells or their derivatives produced or maintained by a method of the invention.

In an eleventh aspect the present invention provides a method of maintaining isolated MM in culture in the absence of a functional ureteric tree which method comprises incubating the MM in the presence of TGF-β2.

In a twelfth aspect the present invention provides a method of maintaining MM in culture in the absence of a functional ureteric tree which method comprises incubating the MM in the presence of serum.

The present invention provides in a thirteenth aspect cells for use in renal cell-based therapy comprising renal mesenchymal cells maintained by a method of the eleventh and twelfth aspects of the invention. In a fourteenth aspect the present invention provides a method for repairing severely diseased kidneys and/or generating de novo renal tissue, nephrons or kidneys which method comprises use of renal mesenchymal cells maintained by a method of the eleventh and twelfth aspects of the invention. In a fifteenth aspect the present invention provides use of TGFβ2 for the expansion of resident renal mesenchymal stem cells that upon subsequent TGF- β2 withdrawal, go on to reinitiate nephrogenesis as a method of treating renal disease.

In a sixteenth aspect the present invention provides use of TGF-β2 for the survival and expansion of adult or embryonic stem cells after they have been induced to a renal progenitor state.

In a seventeenth aspect the present invention provides use of TGF-β2 alone or in combination with other members of the TGF-β superfamily or members of other growth factor families for cartilage repair or generation. In this aspect of the invention the TGF-β2 may be used in combination with matrix, as a paste or a gel or surface coating on a scaffold.

In an eighteenth aspect the present invention provides the use of TGF-β2 as a proliferation and expansion reagent to be applied to stem cells (either adult or embryonic) that have been separately induced to become renal progenitors. This use extends to a use as a reagent for addition to metanephric mesenchyme, and also for the expansion of stem-cell derived mesoderm or renal or cartilage progenitors for subsequent delivery to sites such as kidneys and body joints including knee joints, and cartilaginous structures including the external ear, external and internal nose, larynx, epiglottis, trachea, pulmonary bronchi and costal cartilages.

In a nineteenth aspect the present invention provides a method of turning nephrogenesis off in in vitro metanephric culture which method comprises culturing the metanephroi in the presence of TGF-β2. This aspect of the invention extends- to the use of TGF-β2 in turning nephrogenesis off in in vitro metanephric culture.

In a twentieth aspect the present invention provides a method of turning nephrogenesis on in in vitro metanephric culture which method comprises removing TGF-β2 after culturing the metanephroi in the presence of TGF-β2. This aspect of the invention extends to the use of TGF-β2-free culture media in turning nephrogenesis on in in vitro metanephric culture in which nephrogenesis has been turned off by culture in the presence of TGF-β2.

The present invention extends to the use of TGF-β2 in the manufacture of reagents for use in the methods of the invention. In an embodiment of the invention, addition of exogenous TGF-β2 to whole rat or mouse metanephric organ culture results in the generation of at least two distinct mesenchymal lineages. We have called two of these cell types, Type 1 and Type 2 cells,

Type 1 cells are round, compact cells with a high nuclear/cytoplasmic ratio. These cells are only present if TGF-β2 is added from the beginning of the culture and are always found towards the centre of the explant. Long term culture with

TGF-β2 (9-15 days) enhances survival and/or proliferation of these cells.

Histology suggests that these cells have characteristics of chondroblasts. The

ECM surrounding these cells stains with Alcian Blue (a marker of proteoglycans) and RT-PCR for the chondrocyte specific type II collagen (procollagen Type I IB) is positive.

Type 2 cells are spindle-shaped and found at the periphery of the cultured kidneys in the presumed nephrogenic zone. Culture in the presence of exogenous

TGF-β2 leads to the proliferation of these cells. The TGF-β2 may be a synthetic form or analogue or mimick of this factor.

There is a specific binding region or sequence in the TGF-β2 protein that is critical for binding to the receptors. This binding of ligand (TGF-β2) to the receptor then triggers a signal transduction pathway that results in changes in gene transcription and ultimately cell functions. It is anticipated that this binding sequence alone, or alternatively synthetic mimetics, could trigger the signal transduction pathway in the absence of the full TGF-β2 protein.

Throughout the description and claims of this specification, the word

"comprise" and variations of that word, such as "comprising" and "comprises" are not intended to exclude other additives, steps or integers.

Brief Description of the Accompanying Drawings

Figure 1 - Phase contrast photomicrographs of cultured rat metanephroi (Bar = 100μm). A: Day 0 control ; B: Day 3 control; C: Day 6 control; D: Day 0 50ng/ml TGF- β2 ; E: Day 3 50hg/ml TGF- β2; F: Day 6 50ng/ml TGF- β2; G: Day 9 50ng/ml TGF- β2; H: Day 12 50ng/ml TGF- β2; I: Day 15 50ng/ml TGF- β2.

Figure 2 - Photomicrographs of haematoxylin and eosin stained paraffin sections of rat metanephroi either cultured in control media or media supplemented with TGF- β2 (Bars = 100μm). A, B, C: 3 day control cultures; D, E,

F: 3 day 50ng/ml TGF-β2 cultures; G, H, I: 6 day 50ng/ml TGF-β2 cultures; J, K, L:

9 day 50ng/ml TGF-β2 cultures.

Figure 3 - Photomicrographs of Alcian blue stained paraffin sections of uncultured rat nasal structures and rat metanephroi cultured in control media for 3 days or in media supplemented with 50ng/ml TGF- β2 for 9 days. A, B: Alcian blue staining in the cartilage of the nasal septum; C, D: Control cultured metanephroi showing weak Alcian blue staining in the interstitium; E, F, G: TGF- β2 cultured metanephroi showing weak Alcian blue staining in non-Type 1 cells. Insert (F) shows a low power image of the cultured metanephros with Alcian Blue staining in Type 1 cells which are shown at higher power in (G) (Bar = 50μm)

Figure 4 - Photomicrographs of haematoxylin and eosin stained paraffin sections of rat and mouse metanephroi cultured in media supplemented with 50ng/ml TGF- β2 for 9 days. Rat = A, C; Mouse = B, D. A, B: Type 1 cells: Round shape, centrally located; C, D: Type 2 cells: Spindle-shaped, peripherally located. (Bar = 50μm)

Figure 5 - RT-PCR analysis of mouse metanephroi cultured under various conditions. This figure clearly depicts that three key kidney development genes (Wnt4, GDNF, Pax2) continue to be expressed in whole metanephroi cultured for up to 9 days in control media as well as in whole metanephroi cultured in the presence of exogenous 50ng/ml TGF-β2 for up to 9 days. Upon removal of TGF- β2 expression of these genes persists. This finding suggests that despite the fact that nephrogenesis has ceased in the presence of TGF-^β2, the MM cells that remain may still capable of expressing kidney development genes and may therefore be useful for cell-based therapies of kidney diseases. Figure 6- RT-PCR analysis of Collagen 2 Variants A and B expression in cultured mouse metanephroi. Variant A is expressed in non-cartilaginous foetal tissues; Variant B is expressed specifically in mature chondrocytes. 3Cand 6C are 3 and 6 day control cultures; 3T, 6T and 9T are 3, 6 and 9 day 50ng/ml TGF-β2 cultures. In whole metanephroi grown in the presence of TGF-B2 the expression of Variant B is upregulated over the culture period.

Figure 7 - Photomicrographs of PCNA stained isolated MM cells cultured for 6 days in media supplemented with 50ng/ml TGF- β2. In contrast, no cells were found in MM explants grown in control media for 6 days.

The following examples illustrate some preferred embodiments of the invention. However, it should be understood that the following examples are illustrative only and should not be taken as a restriction on the generality of the invention as described above.

Best Method and other Methods of Carrying Out the Invention

The sequence of morphological events in metanephric development in the mouse and rat is very similar to that in the human. Clearly, the human kidney is larger than that of the mouse and rat, and its gross anatomy appears different, but at a microscopic level, the structure of the kidneys of these three species is remarkably similar, both during development and in the adult. We have learned in recent years that the molecular regulation of kidney development in the mouse and rat is also very similar to that in the human. Studies in the mouse and rat thus generally prove extremely informative of the developmental events in the human.

Formulation of TGF-β2 for use in the present invention can be performed in accordance with standard techniques.

MATERIALS AND METHODS

Animals and dissections

Rats - Sprague-Dawley rats were time-mated, with embryonic day 0 (E0) assigned to the day upon which a vaginal plug was found. At the desired embryonic day (E14.5) pregnant rats were anaesthetised with an intra-peritoneal injection of Nembutal (pentobarbitone sodium - 4.8mg/100g body weight) (Merial Australia, Parramatta). When fully anaesthetised a midline abdominal incision was made and embryos located. For culture, embryos were removed from the uterus, weighed and if in the appropriate weight range (0.12-0.16g), decapitated and placed in a dish filled with phosphate buffered saline (PBS). Metanephroi were located using a dissecting microscope, removed and placed in a dish of Dulbecco's Modified Eagle's Medium (DMEM): Ham's F12 liquid medium (Trace Biosciences, Castle Hill, NSW, Australia) including supplements: 5μg/ml of transferrin (Sigma-Aldrich, Castle Hill, NSW, Australia), 12.9μl/ml L-glutamine (Trace Bioscience), 100μg/ml penicillin (Trace Bioscience) and 100μg/ml streptomycin (Trace Bioscience).

Mice - Hoxb7/GFP transgenic mice (B6 x CBA background) (Srinivas et al. 1999) were overnight mated. At E12.5 (day 0 was defined as the day upon which a vaginal plug was found) pregnant female mice were sacrificed by cervical dislocation. A midline abdominal incision was made and embryos located and removed. Embryos were weighed and only those weighing between 0.06 and 0.08g were used. Embryos were decapitated and metanephroi quickly located using a dissecting microscope, removed and immersed in Dulbecco's Modified Eagle's Medium (DMEM): Ham's F12 liquid medium including supplements: 5μg/ml of transferrin (Sigma-Aldrich), 12.9μl/ml L-glutamine (Trace Bioscience), 100μg/ml penicillin (Trace Bioscience) and 100μg/ml streptomycin (Trace Bioscience).

Whole metanephric organ culture Rats: Rat metanephroi were cultured in 350μl of DMEM/Ham's F12 medium containing 5μg/ml transferrin, 12.9μl/ml glutamine, 100U/ml streptomycin and 100μg/ml penicillin in 24 well plates on transfilter polycarbonate membranes (Costar, NY, USA) . Metanephroi were assigned into four groups. The medium for each group was changed daily and the experiments finished after 1-9 days of culture. Group 1 metanephroi were cultured in the presence of DMEM/Ham's F12 containing supplements and 4mM HCI/0.1% BSA. Group 2, 3 and 4 metanephroi were grown in media containing 1 , 10 or 50ng/ml human recombinant TGF- β2 (R&D Systems, Minneapolis, MN, USA) reconstituted in 4mM HCI/0.1% BSA respectively. Metanephroi were imaged using phase contrast microscopy, then analysed using histology (haematoxylin and eosin), immunohistochemistry (PCNA, Pax-2) and RT-PCR (BF2, BMP4, c-Ret, GDNF, Pax2, Wnt4, WntTb, Wnt11 , WT1 , collagen II isoforms A and B, Sox9, HPRT). Mice: Metanephroi from Hoxb7/GFP (green fluorescent protein) transgenic mice (Srinivas et al. 1999) were used and cultured as above for whole rat metanephroi although the only concentration of TGF- β2 used was 50ng/ml. Culture media was changed every 3 days, with culture periods ranging from 3 to 15 days. Throughout the culture period metanephroi were imaged using phase contrast microscopy. At the completion of culture, a variety of techniques were used to analyse metanephroi including histology (haematoxylin and eosin; Alcian Blue), immunohistochemistry (Pax2, WT-1 , PCNA) and RT-PCR (BF2, BMP4, c- Ret, GDNF, Pax2, Wnt4, Wnt7b, Wnt11 , WT1 , collagen II isoforms A and B, Sox9, HPRT).

Immunostaining for PCNA, Pax2, and WT-1

Rat - PCNA and Pax-2 immunohistochemistry were performed on metanephroi cultured for 1 or 3 days in the presence or absence of 50ng/ml TGF- β2. Sections were dewaxed then incubated in the following solutions, with all incubations carried out at room temperature unless otherwise stated: 0.1 M Phosphate Buffered Saline (PBS) (2 changes, 5 minutes each); 1% Triton in PBS (10 minutes); 0.1 M PBS (2 changes, 5 minutes each); 2% H₂O₂ in Methanol (15 minutes); 0.1 M PBS (2 changes, 5 minutes each); 10% Normal Goat Serum (30 minutes); primary antibody, 10ug/ml anti Pax-2 (Zymed Laboratories San Francisco, CA, USA) or 1 :400 PCNA (Dako Corporation, Carpinteria, CA, USA) , (overnight at 4°C); 0.1M PBS (2 changes, 5 minutes each); secondary antibody, 1 :100 Goat anti Rabbit IgG Biotin Conjugate (Sigma Aldrich) (for Pax-2) or 1 :200 Goat anti Mouse Biotin Conjugate (Sigma Aldrich) (for PCNA) (90 minutes); 0.1 M PBS (2 changes, 5 minutes each); Elite streptavidin/biotin amplification ABC kit (Vector Laboratories inc., Burlingame, CA, USA); 0.1 M PBS (2 changes, 5 minutes each); diaminobenzidine/H2θ2 (1 minute); H2O (5 minutes). The sections were briefly counterstained in haematoxylin prior to dehydrating in ascending concentrations of alcohol to xylene and mounting in DPX. Mouse - Pax-2 and WT-1 immunohistochemistry were performed on metanephroi cultured for 9 days in the presence of 50ng/ml TGF- β2 and metanephroi cultured for 9 days in the presence of 50ng/ml TGF- β2 followed by 3 or 6 days in the absence of TGF- β2. Sections were dewaxed then incubated in the following solutions, with all incubations carried out at room temperature unless otherwise stated: 0.1 M PBS (2 changes, 5 minutes each); 10% Normal Goat Serum (30 minutes); primary antibody, 10ug/ml anti Pax-2 (Zymed Laboratories San Francisco, CA, USA) or 10ug/ml anti WT_1 (Dako Corporation, Carpinteria, CA, USA) (overnight at 4°C); 0.1M PBS (2 changes, 5 minutes each); secondary antibody, 1 :500 Alexa-Fluor 594 Goat anti Rabbit (Molecular Probes, Eugene, OR, USA) (for Pax-2) or 1 :500 Alexa-Fluor 488 Goat anti Mouse (Molecular Probes) (for WT-1) (90 minutes); 0.1 M PBS (2 changes, 5 minutes each) The sections were also stained with Dapi which stains cell nuclei making possible the identification of histological features.

RNA extraction

At the completion of the culture period metanephroi were stored at -70°C. For RNA extraction an RNA extraction kit was used (Qiagen, Clifton Hill, Australia). RNA was run on a mass spectrometer to determine if the RNA was of good quality

Making cDNA from RNA

5μg of total RNA was added to 1μl of 10mM dNTP mix, 1μl of oligo (dT) and 10μl of DEPC-treated water, the reaction incubated at 65°C for 5 min then chilled on ice. A reaction mixture (consisting of 2μl of 10x RT buffer, 4μl of 25mM MgCI₂, 2μl of 0.1M DDT and 1μl RNaseOUT recombinant RNase inhibitor (Invitrogen, Carlsbad, USA)) was added to the RNA mixture and briefly centrifuged, then incubated at 42°C for 2 min. 1μl of SUPERSCRIPT II RT (Invitrogen) was added to the sample tubes only and incubated at 42°C for 50 min. Reactions were stopped at 70°C for 15 min then chilled on ice. The products of these reactions were collected by brief centrifugation. 1μl of RNase H (Invitrogen) was added to the mixture and incubated for 20 min at 37°C.

Polvmerase Chain Reaction (PCR) PCR was used to molecularly characterize metanephroi cultured in the presence or absence of rhTGF- β2. The genes investigated were kidney development genes (BF-2, BMP-4, C-Ret, GDNF, Pax-2, Wnt-4, Wnt-7b, Wnt-11, WT-1), chondrogenic genes (Collagen 2, SOX-9) and a housekeeping gene (HPRT). To a 0.2ml thin walled PCR tube, 2μl of cDNA was added to a mixture containing: 5μl of 10xPCR buffer minus Mg, 1.5 μl of 50mM MgCI₂, 1μl of 10mM dNTP, 1μl of 10μM sense primer, 1μl of 10μM antisense primer, 0.4μl of Taq DNA polymerase (Invitrogen) and 38.1μl of DEPC-treated water giving a final volume of 50μl. These tubes were then incubated at 94°C for 2 min followed by 35 cycles of the following: denaturing (94°C for 30 sec), annealing (8°C below melting temperature (Table 2) for 40 sec), lengthening (72°C for 50 sec). Samples were then left at 72°C for 2 min and chilled to 4°C. Loading dye was added to 20μl of each sample which were electrophoresed in a 1% DNA grade agarose gel. The samples were run against a 1kb DNA ladder. Gels were viewed under an ultraviolet light to visualize gene products.

Culture of isolated MM without UB

In addition to culture of whole mouse metanephroi, culture studies were performed using isolated mouse MM explants. In these studies, the MM was separated from the UB and then the isolated MM was cultured alone in the presence or absence of exogenous TGF-β2 (50ng/ml). Four culture conditions were employed: (1 ) media as for whole metanephric culture above without serum; (2) media as above containing 10% fetal calf serum (Trace Biosciences); (3) media as above without serum and containing 50ng/ml TGF-β2; or (4) media as above containing 10% fetal calf serum and 50ng/ml TGF-β2.

To obtain isolated MM, whole mouse metanephroi were removed as described above at E11.5 (one day earlier than for whole organ culture) and placed in phosphate buffered saline. Metanephroi were then collagenase-treated (0.2% collagenase) (Scimar, Templestowe, Vic, Australia) for 15 minutes at 37°C and the UB was physically separated from the MM using fine dissection needles. Isolated MM explants were cultured on transwell polycarbonate filters for between 3 and 9 days. Media was changed every 3 days. During and at the end of the culture period isolated MMs were imaged using phase contrast microscopy. At the completion of culture, MMs were analysed using histology, DAPI staining and PCNA histochemistry. Results

Morphology of control cultured rat and mouse metanephroi

At the beginning of the culture period rat and mouse whole metanephroi consisted of a T-shaped ureteric tubule and MM After 1 day of culture, metanephroi cultured in control medium showed several branches of the ureteric duct, together with mesenchymal condensates. No S-shaped bodies, comma- shaped bodies or glomeruli were observed at this stage. At 3 days, mesenchymal condensates, comma-shaped bodies and S-shaped bodies were observed, and a few mature glomeruli could also be seen. After 5 or more days, continued growth and differentiation of the metanephros was seen, with many mesenchymal condensates, comma-shaped bodies, S-shaped bodies and glomeruli observed. See Figures 1 and 2A, B, C for representative photomicrographs.

Morphology of rat and mouse metanephroi cultured in the presence of exogenous TGF-β2

The phenotype of whole rat and mouse metanephroi cultured in the presence of exogenous TGF-β2 was strikingly different to that of control metanephroi. Culture in the presence of 50ng/ml TGF-β2 (and 10 and 100ng/ml TGF-β2) lead to a marked suppression of metanephric development and nephrogenesis (Figure 2D-L). By phase contrast microscopy, the TGF-β2 cultured explants appeared small and dark in comparison to explants cultured in the control media (Figure 1 E-I). Histology revealed that the explants contained many cells that appeared to be alive. However, very few nephrons were evident, and the UB was small and unbranched compared to what was seen in control culture. Moreover, the majority of the explant was comprised of morphologically- undifferentiated mesenchymal cells. This phenotype is unlike anything previously seen by us in our studies of TGF-β superfamily members and metanephric development, and to our knowledge has not been previously described in the literature. The phenotype observed was very similar between rat and mouse cultures (Figure 4). Our initial studies focused on the rat, then to enable us to take advantage of molecular approaches we focused on the mouse.

Further histological examination of the explants cultured in the presence of exogenous TGF-β2 revealed at least two distinct mesenchymal cell types. Type 1 cells were always located towards the centre of explants and resembled embryonic cartilage, in that the cells were large and round, and contained a moderate amount of ECM between them. The edge of the Type 1 cell regions resembled perichondrium. Type 1 cells and their surrounding ECM stained with Alcian blue (Figure 3). Only very limited, weak Alcian blue staining was observed in control metanephroi.

At the edge of explants cultured in the presence of exogenous TGF-β2 we identified what we have called Type 2 cells. These cells contained ovoid nuclei, typically comprised a layer of 5-10 cells, and formed a distinct "capsule" to the explants. These cells were located in the so-called nephrogenic zone of the explants, but the layer was much thicker than the normal nephrogenic zone. These cell types were evident following just 3 days of culture with TGF- β2 and were similarly prominent at 9 days. By 15 days, Typel cells predominated, though the Type 2 cells were still present. Culture of rat metanephroi in control media for 3 days followed by culture in the presence of exogenous TGF- β2 for 3 days resulted in the cessation of nephrogenesis. However, Type 2 cells formed in a peripheral "halo" around the explant, approximately 8-10 cells deep. In contrast, culture in the presence of exogenous TGF- β2 for 3 days followed by culture in control medium for 3 days appeared to lead to a restoration of nephrogenesis.

PCNA. Pax2 and WT1 immunohistochemistry on rat and mouse metanephroi cultured in the presence of TGF-β2

Immunohistochemistry for PCNA was performed on 1 and 3 day rat 50ng/ml TGF- β2 treated cultures to demonstrate cell proliferation. After 1 day of culture TGF- β2 cultured metanephroi showed extensive staining. At 3 days of culture TGF- β2 cultures, composed almost solely of mesenchymal cells, showed high levels of proliferation, although not as much as day 1 of the culture period. Immunohistochemistry for Pax-2 was performed on 3 day rat TGF- β2 cultures to determine whether the mass of cells in TGF- β2 treated metanephroi were uninduced mesenchymal cells or cells that had condensed. After 1 day of culture metanephroi showed strong Pax-2 staining in condensing mesenchyme and weaker staining in the epithelial tubules. The mass of mesenchymal cells in 3 day TGF- β2 cultures showed no staining for Pax-2, indicating the presence of mesenchymal cells that had not condensed. Pax-2 and WT-1 staining of mouse TGF-β2 treated cultures showed no positive staining in the mesenchymal cells in 9 day cultures and 9 day TGF-β2 cultures followed by 3 or 6 days in control media. The only positive staining was in the ureteric epithelium and early nephron structures (for Pax-2) and the podocytes (for WT-1 ). These data suggest that mesenchymal cell condensation and epithelialisation (i.e. nephrogenesis) has ceased in the presence of TGF-β2.

RT-PCR analyses of rat and mouse metanephroi cultured in the presence of TGF- β E15 mouse metanephroi and E12.5 mouse metanephroi cultured for 3 days under control conditions were used as positive controls and were found to express mRNAs for all kidney genes (BF2, BMP4, c-Ret, GDNF, Pax2, Wnt4, Wnt7b, Wntl 1 , WT1) tested. E15 rat metanephroi and E14 rat metanephroi cultured for 3 days in control media were used as positive controls. These expressed all kidney genes except Wnt7b and BF2. This is most probably due to the fact that the primers were designed for the mouse genome, and they consequently did not overlap with the rat sequence.

RT-PCR analysis of mouse metanephroi cultured in the presence of TGF- β2 revealed expression of all kidney markers tested (BF2, BMP4, c-Ret, GDNF, Pax2, Wnt4, Wnt7b, Wntl 1 , WT1 ) at 3, 6 and 9 days of culture and in metanephroi grown in TGF-β2 for 9 days followed by 3 or 6 days culture in control media (Figure 5). In rat metanephroi cultured in the presence of TGF-β2 Wnt7b and BF2 were not expressed. Again, this is most probably due to the fact that the primers were designed for the mouse genome, and they consequently did not overlap with the rat sequence. Nonetheless, the data indicate that cells in the explants continue to express many of the key genes involved in kidney development and nephrogenesis, despite the fact that nephrogenesis and normal kidney development has ceased in the presence of exogenous TGF- β2. This suggests that these explants continue to have a generally "renal" molecular profile, but that they are not able to undergo nephrogenesis in the presence of TGF-β2. Thus, following appropriate molecular stimulation, these explants could recommence nephrogenesis and relatively normal kidney development. Expression of collagen type 2B, a specific marker of mature chondrocytes, was found in 3, 6 and 9 day TGF-β2 cultures of whole mouse metanephroi, suggesting that these cells may be chondrocyte progenitor cells or of a chondrocyte lineage (Figure 6).

Culture of isolated MM Survival/non-survival of cultured isolated MM explants was based on phase contrast microscopy and DAPI immunostaining. Of the 14 explants cultured in control media without TGF-β2 and fetal calf serum, surviving cells appeared to be present in just two explants. In contrast, of the 20 explants cultured in media containing 50ng/ml TGF-β2, surviving cells were seen in all 20 explants. Cell survival was also observed in explants cultured in media containing fetal calf serum (9/11 ) and in media containing fetal calf serum and TGF-β2 (9/13).

Histological analysis (haematoxylin and eosin) of control and TGF-β2 cultures revealed that explants cultured in control media (no FCS or TGF-β2) contained no cells. In contrast, explants cultured in the presence of exogenous TGF-β2 contained significant numbers of cells, which demonstrated little morphological differentiation (Figure 7). No epithelial differentiation was observed, and PCNA immunostaining revealed a high proportion of cells undergoing proliferation (Figure 7).

This is the first report of TGF-β2 facilitating survival and proliferation of cells in cultured isolated MM explants, and indicates culture of isolated MM in serum- free conditions in the presence of 50ng/ml TGF-β2 leads to the survival and proliferation of cells derived from the MM, as determined by haematoxylin and eosin and PCNA immunostaining.

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Claims

1. A method for maintaining the survival of a renal progenitor cell population which method comprises treating the population with TGF- β2.

2. A method for expanding a renal progenitor cell population which method comprises treating the population with TGF-β2.

3. Use of TGF-β2 to drive MM cells towards a renal and/or a cartilage phenotype by incubating the MM cells in the presence of TGF-β2.

4. A method for inducing the production of mesenchymal lineages from kidney cells which method comprises treating the kidney cells with TGF- β2.

5. A method according to claim 4 wherein the mesenchymal lineage is a renal or cartilage lineage.

6. A method according to claim 4 wherein incubation in the presence of TGF-β2 from culture initiation promotes formation of renal progenitor cells and cartilage progenitor cells.

7. A method according to claim 4 wherein addition of TGF-β2 at any time promotes formation of renal progenitor cells.

8. A method for expanding and isolating renal and/or cartilage progenitor cells which method comprises culturing the cells in the presence of exogenous TGF-β2 to expand the cells and then isolating the cells by a suitable cell isolation technique.

9. A method according to claim 8 wherein culturing in the presence of TGF-β2 from culture initiation promotes formation of renal progenitor cells and cartilage progenitor cells whereas addition of TGF-β2 at any time promotes formation of renal progenitor cells.

^• 10. A method according to claim 8 wherein expansion of these cells permits their molecular characterisation, identification of specific markers and the use of these markers in isolating separate populations of these cells.

11. Use of TGF-β2 treated mesenchymal cells as a source of renal or cartilage progenitor cells.

12. Use of the renal lineage cells according to claim 5 in repairing diseased kidneys and/or generating de novo renal tissue, including nephrons, vasculature and interstitium, or whole kidneys for patients with kidney disease.

13. Use of the cartilage lineage cells according to claim 5 in growing in vitro cartilage segments or prostheses for transplantation into patients, or alternatively for cell-based therapy for patients with cartilage defects.

14. Cells for use in renal cell-based therapy comprising renal progenitor cells or their derivatives produced or maintained by a method according to claim 1 or claim 2.

15. A method for repairing severely diseased kidneys and/or generating de novo renal tissue, nephrons or kidneys which method comprises use of renal progenitor cells produced or maintained by a method according to claim 1 or claim 2.

16. Cartilage mesenchymal cells produced or maintained by a method according to claim 5 wherein TGF-β2 is present from the beginning of the culture, for use in growing cartilage prostheses in vitro for in vivo applications, and/ or cell-based therapy of cartilage defects in vivo.

17. A method of cartilage repair comprising use of cartilage cells or their derivatives produced or maintained by a method according to claim 5.

18. A method of turning nephrogenesis off in in vitro metanephric culture which method comprises culturing the metanephroi in the presence of

TGF-β2.

19. Use of TGF-β2 in turning nephrogenesis off in in vitro metanephric culture.

20. A method of turning nephrogenesis on in in vitro metanephroi culture which method comprises removing TGF-β2 after culturing the metanephroi in the presence of TGF-β2.

21. Use of TGF-β2-free culture media in turning nephrogenesis on in in vitro metanephric culture in which nephrogenesis has been turned off by culture in the presence of TGF-β2.

22. A method of maintaining MM in culture in the absence of a functional ureteric tree which method comprises incubating the MM in the presence of TGF-β2.

23. A method of maintaining MM in culture in the absence of a functional ureteric tree which method comprises incubating the MM in the presence of serum.

24. Cells for use in renal cell-based therapy comprising renal mesenchymal cells maintained by a method according to claim 22 or 23.

25. A method for repairing severely diseased kidneys and/or generating de novo renal tissue, nephrons or renal vasculature, interstitium or whole kidneys which method comprises use of renal mesenchymal cells maintained by a method according to claim 22 or 23.

26. Use of TGFb2 for the expansion of resident renal mesenchymal stem cells that upon subsequent TGF-β2 withdrawal, go on to reinitiate nephrogenesis as a method of treating renal disease.

27. Use of TGF-β2 for the survival and expansion of adult or embryonic stem cells after they have been induced to a renal progenitor state.

28. Use of TGF-β2 alone or in combination with other members of the TGF- β superfamily or members of other growth factor families for cartilage repair or generation.

29. Use according to claim 29 wherein the TGF-β2 is used in combination with matrix, as a paste or a gel.

30. Use of TGF-β2 as a proliferation and expansion reagent to be applied to embryonic or adult stem cells that have been separately induced to become renal progenitors.

31. Use according to claim 30 wherein the TGF-β2 is used as a reagent for addition to metanephric mesenchyme.

32. Use according to claim 30 wherein the TGF-β2 is used as a reagent for expansion of stem-cell derived mesoderm or renal or cartilage progenitors for subsequent delivery to sites such as kidneys and body joints including knee joints.

33. Use of TGF-β2 in the manufacture of a reagent for use in a method according to any one of claims 1 ,2,4-10, 15,17,18,20,22,23 and 25.