WO2007079533A1 - Method for establishing and proliferating human esc by co-culturing with allogeneic feeder cells in serum-free media - Google Patents

Abstract

Described herein is the establishment of a human embryonic stem cell line designated Endeavour- 1 under serum free and substantially allogeneic conditions. Also described is the production of 4 clonal cell lines, designated E1C1, E1C2, E1C3 and E1C4, which are derived from Endeavour-1. Each of these cells lines demonstrates pluripotency and long-term self renewal, and the ability to be directed to differentiate into cells exhibiting characteristics of any one or more of ectoderm, mesoderm and endoderm. Also described are cells derived from these cell lines, differentiated cells derived from these lines and uses thereof, Also described are methods of establishing and proliferating a human embryonic stem cells line in an undifferentiated form under serum-free and substantially allogeneic conditions.

Description

Method for establishing and proliferating human ESC by co-culturing with allogeneic feeder cells serum-free media

Technical Field

The present invention relates to embryonic stem cells. In particular, the present invention relates to new human embryonic stem cell lines, methods of establishing, proliferating and differentiating the cell lines and uses thereof.

Related Applications

This application claims priority to Australian provisional patent application 2006900110 which was filed 10 January 2006, Australian patent application 2006202652 which was filed 22 June 2006, US patent application 11/474,059 which was filed 22 June 2006 and Australian patent application 2006213942 which was filed 1 September 2006, the entire contents of each of which is incorporated herein by cross reference.

Background of the Invention

Stem cells are distinguishable from other cell types in that they are capable of both differentiating into specialized cells and dividing continuously for long periods of time, thus making them suitable as cell lines in research. They are found in embryonic, fetal and adult tissues.

Cells of a human embryo up to the 8 cell stage are "totipotent", each cell being capable of developing into an entire human being. As the cells of an embryo continue to divide, they form a blastocyst, being a hollow sphere of about 120 cells with an outer layer and an inner cell mass. The outer layer develops into the placenta while the inner cell mass comprises embryonic stem cells (ESCs) which are "pluripotent", being capable of differentiating into all cell types found in a human body. However, as pluripotent ESCs cannot develop into tissues necessary to support pregnancy, such as the placenta, ESCs cannot of themselves develop into a human being.

Following the successful derivation of five human embryonic stem cell (hESC) lines in 1998, many new hESC lines have been created worldwide. To date, it is estimated that more than 220 new hESC lines have been produced world-wide, of which about 78 are currently listed in the National Institute Health (NIH) registry. Of these 78 lines, only about 26 have been characterized to varying degrees and are available for research. In addition, many of these hESC lines are not clonal, were derived under different culture conditions and were propagated on different feeder layers such as mouse embryonic fibroblasts (MEF), STO (an immortal mouse embryonic fibroblast cell line), fetal muscle, skin, foreskin and adult fallopian tube epithelial cells. Moreover, the culture of some of these lines involves feeder free and/or serum free systems, therefore making comparison between lines very difficult. Differences in gene expression have also been reported in some of these lines. Recent attempts to derive hESCs under serum-free defined conditions have relied on

"immunosurgery" to isolate the inner cell mass and have utilised fetal calf serum to generate the feeder cells lines which maintain the hESC lines. Attempts to minimise the use of xenogenic sera, for example through the use of human serum, tend to cause the differentiation of hESCs in long- term culture.

During the last 5 years, there has been an emphasis in the scientific community on improving hESC culture conditions, undertaking hESC genetic manipulations and optimizing differentiation protocols to produce progeny for transplantation and drug testing. Recent breakthroughs using somatic cell nuclear transfer (SCNT) in human lines, nuclear reprogramming by fusions and creation of SCNT lines may eventually result in custom-made therapies. However, in attempting to achieve these goals, the scientific community remains seriously limited by the lack of optimized protocols to obtain relatively pure populations of specified lineages from hESC lines using current in vitro culture conditions and procedures. This may be due to a lack of quality controls and initial variability (or lack of uniformity) in these hESC lines. Indeed, only a handful of studies have examined these parameters in order to attempt to achieve uniformity in lineage selections.

The present inventors have now developed a novel human embryonic stem cell line, designated Endeavour-1 (E1) and four clonal cell lines derived from E1, from donated human embryos, in serum-free conditions on a feeder layer of human fetal fibroblasts.

Summary of the Invention

According to a first aspect, there is provided a human embryonic stem cell (hESC) line or an isolated human embryonic stem cell thereof selected from the group consisting of: an hESC line designated Endeavour-1 wherein the hESC line has been deposited pursuant to the Budapest Treaty with the China Centre for Type Culture Collection (CCTCC) on 6 January 2006 under Accession Number C200602, an hESC line designated E1C1, wherein the hESC line has been deposited pursuant to the Budapest Treaty with the China Centre for Type Culture Collection (CCTCC) on 8 August 2006 under Accession Number C200626, an hESC line designated E1C2, wherein the hESC line has been deposited pursuant to the Budapest Treaty with the China Centre for Type Culture Collection (CCTCC) on 8 August 2006 under Accession Number C200627, and an hESC line designated E1C4, wherein the hESC line has been deposited pursuant to the Budapest Treaty with the China Centre for Type Culture Collection (CCTCC) on 8 August 2006 under Accession Number C200628. In one embodiment of the first aspect, cells of the hESC line display any one or more of the characteristics selected from the group consisting of: (a) pluripotency,

(b) long-term self renewal,

(c) the expression of any one or both of the stage-specific embryonic antigens (SSEAs) SSEA-3, and SSEA-4, (d) the expression of any one or both of the tumor recognition antigens (TRAs) TRA-1-60 and TRA-1-81,

(e) the expression of 0CT4,

(f) the intracellular expression of alkaline phosphatase (ALP),

(g) the expression of Nanog, (h) embryoid body formation in suspension culture conditions, and

(i) teratoma formation after implantation of cells of the hESC line beneath the kidney capsule of NOD-SCID mice, the teratoma comprising differentiated cells and tissues expressing characteristics representing each of endoderm, mesoderm and ectoderm. According to a further aspect there is provided a hESC line or an isolated hESC cell derived from Endeavour-1 or its clonal cell lines E1C1, E1C2, or E1C4. The derived hESC line or isolated hESC may be a clone.

According to another aspect, there is provided a differentiated cell derived from a hESC line of one of the above aspects. The differentiated cell may be an adult or tissue stem cell. The differentiated cell may exhibit characteristics of endoderm, mesoderm or ectoderm. The differentiated cell may exhibit characteristics of a vascular cell, a heart cell, a nerve cell, a lung cell, a kidney cell, a liver cell, a spleen cell, an epithelial cell or a pancreatic cell. The differentiated cell may exhibit characteristics of a pancreatic cell. The differentiated cell exhibiting characteristics of a pancreatic cell may be an insulin-producing cell. The differentiated cell may be a ceil of neural lineage, According to another aspect, there is provided a method for producing a differentiated cell from a hESC line of one of the above aspects, wherein said method comprises co-culturing cells of the hESC line with feeder cells, contacting the hESC cells with one or more differentiation factors, and culturing the cells under conditions suitable to induce differentiation of the cell, Optionally, the method may further comprise screening cells for characteristics of the differentiated cell, and optionally separating substantially differentiated cells from undifferentiated cells. The method may also comprise genetic manipulation of the hESC cells. According to another aspect, there is provided a differentiated cell produced by the method described herein,

According to a further aspect, there is provided a method of treating a disease in a subject, wherein said method comprises administering to the subject a differentiated cell derived from a hESC of one of the above aspects, The disease may be diabetes. According to a further aspect, there is provided a pharmaceutical composition comprising hESC of one of the above aspects or cells derived from hESC of one of the above aspects, together with a pharmaceutically acceptable carrier, diluent or excipient,

According to yet another aspect there is provided use of a differentiated cell derived from a hESC of one of the above aspects in the manufacture of a medicament for the treatment of a disease.

According to another aspect, there is provided a method for establishing and proliferating cells of a hESC line in an undifferentiated form, wherein said method comprises co-culturing the cells with feeder cells in serum free and substantially allogeneic conditions, The feeder cells may be human embryonic fibroblast feeder cells. The feeder cells may be genetically modified. In one embodiment, the hESC cell line is a cell line of one of the aspects described above.

Definitions

In the context of this specification, the term "comprising" means will be understood to imply the inclusion of a stated step or element or integer or group of stems or elements or integers , but not the exclusion of any other step or element or integer or group thereof. Thus, in the context of this specification comprising means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings. Throughout this specification, reference to "a" or "one" element does not exclude the plural, unless context determines otherwise. For instance, reference to "a hESC" should not be read as excluding the possibility of multiple hESC.

The term "expression" as used herein refers interchangeably to expression of a gene or gene product, including mRNA and the encoded protein. The term "differentiation factor" as used herein refers to any molecule or compound, natural or synthetic, capable of inducing or promoting the differentiation of a pluripotent cell into a specialized form. Methods for determining whether cells have become more differentiated, such as in response to exposure to a differentiation factor, are known and are described, for example, in US patent serial number 7,153,650 (the entire contents of which are incorporated herein by reference). hESC cells or cell lines are described as "undifferentiated" when a substantial proportion (in certain embodiments at least 20%, at least 30%, at least 40%, at least 50%, at least 80% or at least 90%) of stem cells in the population display morphological characteristics of undifferentiated cells, distinguishing them from differentiated cells of embryo origin. It is understood that colonies of undifferentiated cells within the population will often be surrounded by neighbouring cells that are differentiated. It is also understood that the proportion of cells displaying the undifferentiated phenotype will fluctuate as the cells proliferate and are passaged from one culture to another. Cells are recognized as proliferating in an undifferentiated state when they go through at least 4 passages and/or 8 population doublings while retaining at least about 40%, at least about 50%, at least about 60% or the same proportion of cells bearing characteristic markers or morphological characteristics of undifferentiated cells. A "differentiated cell" is a cell that has progressed down a developmental pathway, and includes lineage-committed stem or progenitor cells, such as "adult" or tissue stem cells, and terminally differentiated cells.

The term "embryoid bodies" refers to aggregates of differentiated and undifferentiated cells that appear when pluripotent stem cells overgrow in monolayer cultures, or are maintained in suspension cultures. Embryoid bodies are a mixture of different cell types, typically from several germ layers, which are distinguishable by morphological criteria and by cell markers identified using immunocytochemistry.

"Feeder cells" or "feeder layers" are terms used to describe cells of one type that are co- cultured with cells of another type, to provide an environment in which the cells of the second type can grow.

A cell which is "derived" from an original cell is an immediate or distant progeny of the original cell. The derived cell may be genetically and/or phenotypically identical to the original cell, or it may genetically and/or phenotypically different. Typically but not exclusively a derived cell is more differentiated than the original cell. For example, a cell derived from an ESC may be a stem cell which is committed to the production of cells of a specific lineage. A cell which is derived from an original cell may arise as the result of one or more cell divisions, and/or as a result of genetic manipulation of the original cell or its progeny and/or as a result of nuclear transfer or cell fusion.

A cell which "exhibits or expresses characteristics of a cell of a particular class or type where it exhibits one or more phenotypic traits which are highly suggestive or definitive of cells of that class or type. For example, a cell which exhibits characteristics of a cell of endoderm germ layer origin may exhibit α-fetoprotein expression, or the expression of other markers or trait, such as the formation of ciliated or mucous-producing epithelial layers, which are useful for identifying cells of endoderm origin in contrast to ectoderm or mesoderm germ layer origin. A cell which exhibits characteristics of a cell of the mesoderm cell layer may for example express the markers renin or brachyury, or markers specific for connective tissue, muscle, the vascular system or the urogenital system. A cell which exhibits characteristics of a cell of ectoderm germ layer origin may, for example, express nestin.

In the context of this specification, the term "treatment" refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of a disease, or otherwise prevent, hinder, retard or reverse the progression of disease or other undesirable symptoms in any way. Any description of prior art documents herein, or statements herein derived from or based on those documents, is not an admission that the documents or derived statements are part of the common general knowledge of the relevant art in Australia or elsewhere,

For convenient reference, the following abbreviations are used throughout this specification:

ALP alkaline phosphatase

AFP α-fetoprotein

E1 Endeavour-1

EB embryoid body

ESCs Embryonic Stem Cells hESCs human Embryonic Stem Cells

HFF human fetal fibroblasts

ICM inner cell mass

P passage

PBS phosphate buffered saline

RT-PCR reverse transcriptase polymerase chain reaction

SR serum replacer

SSEA stage-specific embryonic antigen

TRA tumour recognition antigen

Brief Description of the Drawings

Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings:

Figure 1. This figure illustrates the production of differentiated cells in teratomas following injection of Endeavour-1 cells beneath the kidney capsule of NOD-SCID mice. Pluripotency is indicated by the formation of tissues derived from each of the three germ layers, (A) ectoderm, (B) endoderm, and (C) mesoderm, visualized in haematoxylin-eosin stained paraffin sections of teratomas.

Figure 2. This figure illustrates the in vitro differentiation of Endeavour-1, P12 (E1, passage 12) after embryoid body formation. The upper panel demonstrates the derivation of cells of neuronal (ectodermal) lineage: left, nuclear staining with DAPI, middle, immunolocalisation of β-lll tubulin (marker for neuronal lineage) and right, overlap of left and middle images. The lower panel- demonstrates the derivation of cells of endodermal origin by the immunolocalisation of the endodermal marker, α-fetoprotein: with DAPl staining (left), α-fetoprotein staining (middle) and overlap image (right). Figure 3. This figure illustrates the expression of Nanog, which is associated with pluripotency, and the expression of various markers of differentiation, including α-fetoprotein (AFP, a marker of endoderm), GATA4 (a definitive endoderm marker), Nestin (a marker of ectoderm) and Renin (a marker of mesoderm) in Endeavour-1 and its clonal lines, E1C1, E1C2, E1C3 and E1C4 as detected by RT-PCR. The expression of β-actin was used as a control for normalising the expression measurements. The column marked "M" shows molecular weight markers.

Figure 4. This figure demonstrates the results of karyotype analysis of Endeavour-1 at passage 19 and its clonal cell lines E1C1, E1C2, E1C3 and E1C4 (all at passage 4). The karyotype of E1 C3 P4 shows translocation. Figure 5. This figure illustrates the expression of stem cell surface markers SSEA-4, TRA-1-

60 and TRA-1-81 by the clonal cell lines of Endeavour-1, E1C1, E1C2, E1C3 and E1C4 as identified by immunohistochemistry.

Figure 6. This figure illustrates semi-quantitative analyses of β-lll Tubulin (top graph), AFP (α-fetoprotein, middle graph), and CD34 (bottom graph) immunolabeling as markers for ectoderm, endoderm and mesoderm origin respectively, in differentiated cultures of Endeavour-1, E1C1, E1C2, E1C3, and E1C4 (columns from left to right respectively). The Y-axis of each graph refers to the % cells which were positive for the immunolabel.

Figure 7. This figure illustrates the effect of activin A treatment on the expression of the definitive endoderm marker genes FOXa2, GATA4 and Sox17; the pluripotent marker genes Nanog and OCT-4; the mesoderm marker brachyury; and the ectoderm marker, Nestin as identified by RT- PCR in E1 and its clonal lines E1C1, E1C2, E1C3,and E1C4.

Figure 8. This figure illustrates a quantitative analysis of the effect of activin A treatment on the expression of the definitive endoderm marker genes FOXa2, GAT A4 and Sox17; the pluripotent marker genes Nanog and OCT-4; the mesoderm marker brachyury; and the ectoderm marker, Nestin as identified by RT-PCR in E1 and its clonal lines E1C1, E1C2, E1C3, and E1C4. The data is expressed as mean ± standard error mean (SEM). For each group n = 3, student's t test were analysed on clones individually comparing treated and non-treated. *p < 0.05, represents a significant difference in each individual clone between treatment.

Figure 9. This figure illustrates the formation of differentiated cells in teratomas from the clonal cell lines E1C1, E1C3 and E1C4 (each at passage 4) as identified in haematoxylin-eosin- stained paraffin sections. E1C2 at passage 4 also produced similar teratomas. The left hand column of photos illustrates sections through teratomas showing various structures (arrows), the column of photos second from the left shows intestine-like structures (endoderm); the column of photos second from the right shows cartilage-like structures (mesoderm); and the right hand column of photos shows neuroectoderm-like structures (ectoderm). Detailed Description

Pluripotent embryonic stem cells (ESCs) are capable of both differentiating into specialized cells of numerous lineages and long-term self renewal (dividing continuously for long periods of time to produce new pluripotent embryonic stem cells). They can be defined using various established criteria, and characteristically display particular cell surface antigens including the stage-specific embryonic antigens (SSEAs), SSEA -3, SSEA-4, the tumor recognition antigens (TRAs) TRA-1-60 and TRA-1-81, the pluripotent mRNA markers Nanog and the POU-domain transcription factor OCT-4, The pluripotent intracellular marker, alkaline phosphatase, is also indicative of ESCs. In addition, ESCs characteristically express particular differentiation markers for ectoderm (Nestin), mesoderm (Renin) and endoderm (α-fetoprotein and GATA6) on differentiation. Furthermore, embryoid body (EB) formation can be observed in suspension cultures, with EBs characteristically differentiating into a variety of cell types in vitro, thereby indicating pluripotency. To assess pluripotency in vivo, ESCs can be injected beneath kidney capsules of NOD-SCID mice and their ability to form teratomas which comprise cells of different lineages may be assessed.

As disclosed herein the present inventors have successfully derived a novel human embryonic stem cell line (hESC), designated Endeavour-1, from donated^' human embryos, under serum-free conditions and on a feeder layer derived from human fetal tissue. The inventors have found that Endeavour-1 cells display immunohistochemical localisation of alkaline phosphatase and the expression of the Nanog gene as indicated by RT-PCR, each of which are indicative of pluripotency. In addition, embryoid body formation was observed in suspension cultures, and after seeding, EBs were observed to differentiate into various cell types which express characteristics of ectoderm, mesoderm and endoderm in vitro, thereby further indicating pluripotency. Moreover, the inventors have successfully freeze-thawed and passaged the Endeavour-1 cell line at least 15 times, providing further evidence of pluripotency and immortality.

Accordingly, one aspect of the present invention relates to a human embryonic stem cell (hESC) line referred to herein as "Endeavour-1" or Ε1", wherein the hESC line has been deposited pursuant to the Budapest Treaty with the China Centre for Type Culture Collection (CCTCC) on 6 January 2006 under Accession Number C200602. The inventors have also cloned cells present in the Endeavour-1 cell line to produce four new clonal cell lines E1C1, E1C2, E1C3 and E1C4. Of these, the clonal cell line E1C1 has been deposited pursuant to the Budapest Treaty with the China Centre for Type Culture Collection (CCTCC) on 8 August 2006 under Accession Number C200626, the clonal cell line E1C2 has been deposited pursuant to the Budapest Treaty with the China Centre for Type Culture Collection (CCTCC) on 8 August 2006 under Accession Number C200627 and the clonal cell line E1C4 has been deposited pursuant to the Budapest Treaty with the China Centre for Type Culture Collection (CCTCC) on 8 August 2006 under Accession Number C200628.

The hESC lines described herein exhibited spontaneous or directed differentiation under appropriate conditions into cells of all three germ cell layers, the ectoderm, mesoderm and endoderm, and accordingly possess the potential to produce cells of any tissue of the body. Persons of skill in the art will further appreciate that the undifferentiated embryonic stem cells of Endeavour-1 or its clonal cell lines, or pluripotent or multipotent cells derived from these cell lines may be induced in similar ways to other stem cells to differentiate into particular cell types by the exposure of these cells to particular molecules, such as retinoic acid, Writ, Sonic Hedgehog (for inducing neural ectoderm differentiation) and activin A (for inducing endodermal differentiation) or combinations thereof. For example, exposing undifferentiated embryonic stem cells to a combination of retinoic acid, Wnt and Sonic Hedgehog induces the production of neuronal cells, particularly motor neurons (U-Ming, Sidhu & Tuch et ah, 2006; Current Neurovascular Research 3:281-288, the entire contents of which are incorporated herein by reference), and treatment with activin A and retinoic acid produces insulin-producing cells (D'Amour et a/., 2006; Nature Biotechnology 24, 1392-401, the entire contents of which are incorporated herein by reference). In particular embodiments the differentiated cell is a vascular cell, a heart cell, a nerve cell, a lung cell, a kidney cell, a liver cell, a spleen cell, an epithelial cell or a pancreatic cell.

Accordingly, the present invention provides for methods of producing differentiated cells from Endeavour-1 or its clonal cell lines. In one embodiment, the method comprises co-culturing E1, E1C1, E1C2 or E1C4 hESCs with feeder cells, contacting the hESCs with one or more differentiation factors and culturing the cells under conditions suitable to induce differentiation of the hESCs. In another embodiment, co-culture with feeder cells is not required, for example where a defined medium is used to support the survival and differentiation of the hESCs. Factors which may be used to direct the differentiation of embryonic stem cells into cells of one or more of a variety of lineages are reviewed for example in Keller (2005) Genes & Dev 19:1129-1155; Odorico et a/ (2001) Stem Cells 19:193-204; and Trounson (2006) Endocr Rev 27: 208-219, the entire contents of each of which are incorporated herein by reference.

Optionally, the methods may further comprise screening the differentiated cells for characteristics of the differentiated cell. Screening for differentiated cells may be carried out using techniques available in the art. The use of flow cytometry in combination with labelled antibodies directed to surface markers for the differentiated cells of interest is a particularly convenient method to achieve such a screening.

The methods may further comprise separating substantially the differentiated cells from undifferentiated cells or clones. The use of fluorescence activated cell sorting in combination with labelled antibodies directed to surface markers for the differentiated cells and/or stem cells of interest is a particularly convenient method to separate populations of differentiated and undifferentiated cells (see for example Wernig ef a/., 2004; J Neurosci 24, 5258-68, the contents of which are incorporated herein by reference). Similarly, the use of affinity techniques, such as antibody-mediated capture techniques to selective retain cells which express surface labels which are specific for the differentiated cell of interest are well known in the art, and may be used to substantially separate populations of differentiated and undifferentiated cells. The present invention moreover provides differentiated cells or undifferentiated cells produced by the methods as described above.

A person of skill in the art will recognise that all cells of the body are derived from any one of the three germ cell layers in a defined and predictable manner. By manipulating culturing conditions, cells of the hESC cell lines of the present invention can be induced to differentiate into cells which express characteristics of a cell from any one of endodermal, mesodermal or ectodermal germ cell layers, or a mixture of these cell types. Techniques and methodologies for such manipulation will be known to those skilled in the art. This pluripotent capacity of cells of the invention may be utilised, for example, for the generation of cells producing a desired biomolecule. Further, differentiated cells, tissues or organs, the products of cells of the present invention, may also be used, for example, for therapeutic or prophylactic transplantation purposes, or for a range of scientific purposes such as the identification of gene targets for pharmacological agents, for generating transgenic or chimeric organisms to serve as, for example, models of specific human genetic diseases, for studying differentiation, development or other biological processes.

The range of applications of cells of the present application is in no way to be limited by the above discussion. Those skilled in the art will readily appreciate the diversity of applications of embryonic stem cells of the invention, or their progeny.

By way of example only, skilled artisans will appreciate that such differentiated cells may be used for a wide variety of therapeutic applications for humans. Accordingly, the present invention provides methods for treating diseases, comprising administering to a person in need the differentiated cells described above. By way of example, human embryonic stem cell clones of the present invention may be induced to differentiate into insulin-producing cells which in turn may find application in the treatment of diabetes (D'Amour ef a/., 2006 supra; Lumelski ef a/. 2001 Science 292: 1389-1394, the entire contents of which are incorporated by reference). Other potential applications are discussed, for example, in Keller (2005, supra). Particularly contemplated are disorders or diseases involving the death of cells of neural origin, such as in spinal cord injury (U- Miπg, Sidhu and Tuch ef a/., 2006; Current Neurovascular Research 3, 281-288, the entire contents of which are incorporated by reference), Parkinson's disease and Alzheimer's disease (Wernig ef a/., 2004, supra), in which hESCs may be used as a self-renewing source of neurons suitable for implantation to the subject suffering the disease or disorder. The Endeavour-1 cell line was isolated and propagated using serum-free and substantially allogeneic conditions. Accordingly, also provided is a method of establishing and propagating a hESC line using serum free and substantially allogeneic conditions. In one embodiment, the method involves the use of human feeder cells which have been isolated and grown in serum-free defined conditions and preferably established and subcultured using non-xenogeneic proteases. In one embodiment, the serum-free defined conditions comprise high glucose knockout medium supplemented with knockout serum replacer and basic fibroblast growth factor. Although any feeder cell isolated and grown under these conditions and which can support the proliferation of hESCs may be used, in an advantageous embodiment the feeder cells are primary fetal fibroblasts, for example fetal skin fibroblasts. Cells from primary or established human cell lines, such as newborn foreskin fibroblasts which have been established in serum-free, and preferably in allogeneic conditions may also be used as feeder cells

Where the method utilises the isolation of cells from the inner cell mass of a blastocyst, the method may advantageously utilize physical techniques such as laser dissection to remove the inner cells mass from the other cells of the blastocyst. Thie use of animal-derived antibodies of "immunosurgery" to isolate cells of the ICM, or the use of xenogenic sera to support the growth of feeder cells and or hESCs exposes these cells to xenogenic material and potentially contaminates the cell line with animal-derived products, animal-borne viruses or prions.

In addition to the establishment of feeder cells in serum free conditions, in advantageous embodiments the hESCs are cultured in serum free conditions. The culture conditions provided by the methods described herein have exhibited the ability to maintain and proliferate hESCs for extended periods of time without stimulating differentiation It is presently considered that hESC derived in the absence of feeder layers become chromosomally unstable, and so the use of feeder cells is desirable hESCs or cells derived from HESCs of the present invention may be administered as compositions either therapeutically or preventively. In a therapeutic application, compositions are administered to a patient already suffering from a disease, in an amount sufficient to cure or at least partially arrest the disease and its complications. The composition should provide a quantity of the compound or agent sufficient to effectively treat the patient. The therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the compound or agent employed; the composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of sequestration of the agent or compound; the duration of the treatment; drugs used in combination or coincidental with the treatment, together with other related factors well known in medicine. One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of agent or compound which would be required to treat applicable diseases. Generally, an effective dosage is expected to be in the range of about 0.0001 mg to about IOOOmg per kg body weight per 24 hours; typically, about 0.001 mg to about 750mg per kg body weight per 24 hours; about 0.01mg to about 500mg per kg body weight per 24 hours; about 0.1 mg to about 500mg per kg body weight per 24 hours; about 0.1 mg to about 250mg per kg body weight per 24 hours; about 1.Omg to about 250mg per kg body weight per 24 hours. More typically, an effective dose range is expected to be in the range about 1.Omg to about 200mg per kg body weight per 24 hours; about 1.Omg to about 10Omg per kg body weight per 24 hours; about 1.Omg to about 50mg per kg body weight per 24 hours; about 1.Omg to about 25mg per kg body weight per 24 hours; about δ.Omg to about 50mg per kg body weight per 24 hours; about δ.Omg to about 20mg per kg body weight per 24 hours; about δ.Omg to about 1δmg per kg body weight per 24 hours.

Alternatively, an effective dosage may be up to about δ00mg/m². Generally, an effective dosage is expected to be in the range of about 2δ to about 500mg/m², preferably about 2δ to about 3δ0mg/m², more preferably about 25 to about 300mg/m², still more preferably about 2δ to about 2δ0mg/m², even more preferably about δO to about 2δ0mg/m², and still even more preferably about 7δ to about 1δ0mg/m².

Typically, in therapeutic applications, the treatment would be for the duration of the disease state. It will be apparent to one of ordinary skill in the art that the optimal course of treatment, such as, the number of doses of the composition given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.

In the context of pharmaceutical compositions which comprise the new hESC described herein, or cells derived from hESC described herein, in general, suitable compositions may be prepared according to methods which are known to those of ordinary skill in the art and accordingly may include a pharmaceutically acceptable carrier, diluent and/or adjuvant.

These compositions can be administered by standard routes. In general, the compositions may be administered by the parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular), oral or topical route. More preferably administration is by the parenteral route. The carriers, diluents and adjuvants must be "acceptable" in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof.

Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly, Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

The compositions of the invention may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets, elixirs, for example), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection. For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol.

Some examples of suitable carriers, diluents, excipients and adjuvants for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin. In addition these oral formulations may contain suitable flavouring and colourings agents. When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate which delay disintegration.

Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents.

Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents. Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include com starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof,

Suspensions for oral administration may further comprise dispersing agents and/or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate, polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate and the like. The emulsions for oral administration may further comprise one or more emulsifying agents.

Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.

Methods for preparing parenterally administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa., hereby incorporated by reference herein.

The present invention will now be further described in greater detail by reference to the following specific examples, which should not be construed as in any way limiting the scope of the invention.

Examples

Example 1. General Methods

All reagents including culture media and sera were obtained from Gibco/lnvitrogen (Carlsbad, CA USA, http://www.invitrogen.com). hESC colonies were maintained in gelatin-coated six well culture plates (Becton Dickinson, NJ, USA; http://www.bdbiosciences.com) on gamma- irradiated (45 Gy) human fetal fibroblast (HFF; passage 6) feeder layers (1.5 x 10⁶ cells/ml) and cultured at 37⁰C₁5% CO₂ in serum replacer (SR) medium as described below.

Primary serum-free Human Fetal Fibroblasts (HFF) were derived from human fetal skin after therapeutic termination of pregnancies after obtaining maternal consent, Briefly, two 3 mm² pieces of skin were washed twice with PBS containing 25 U/ml penicillin and 25 μg/ml streptomycin and finally chopped into small pieces with a pair of fine scissors. Single cell suspensions were prepared by treating for 15 min at 37⁰C with a xeno-free protease TripLE Select (Gibco/lnvitrogen, Carlsbad, CA USA). After washing, the HFF were cultured in T75 tissue culture flasks coated with human collagen IV (Sigma, MO USA) in SR medium consisting of Dulbecco knockout (KO-DMEM) high glucose, supplemented with 20% knockout serum replacer (Gibco, Carlsbad, CA USA), 2 mM L- glutamine, 0.1 mM nonessential amino acids, 0,01 mM 2-mercaptoethanol, 1x insulin-transferrin- selenium, basic fibroblast growth factor (bFGF) 4 ng/ml, 25 U/ml penicillin and 25 μg/ml streptomycin.

The primary cultures of HFFs were cryopreserved by a standard slow freezing procedure in 10% DMSO.

The serum-free HFFs were validated for supporting the undifferentiated growth of an existing line, hES 3 (ESI Singapore) for many passages. The serum-free HFF produced using the methods described above showed similar morphology to a typical HFF grown in serum-containing medium, and could be used to maintain the undifferentiated growth of the hESC line hES 3 for several passages.

Example 2. Derivation of a new hESC line Endeavour-1 (E1) from donated human embryos

A batch of eight human embryos (morulae) were thawed out for each experiment and cultured in human embryo culture medium (Cook IVF₁ Mile Plain, Australia) in 4-well plates for 3 to 4 days at 37⁰C for development to blastocyst stage. The resulting blastocysts (about 40%) were further cultured for 1 to 2 days to induce blastocyst hatching from zona pellucida. Blastocysts which did not hatch automatically were mechanically hatched by laser breaching of zona pellucida. The hatched blastocysts were cultured onto a serum-free HFF feeder layer in six-well plates in SR medium for 2 to 3 days. The visible inner cell masses (ICM) were dissected out physically by laser and transferred into fresh feeder plates. hESC colonies were observed after two weeks of culture of the ICM. The hESC line, Endeavour-1 (E1) was derived from one of the ICM. The newly emerged hESC colonies were initially passaged 2 to 3 times by physical dissection before using enzymatic dissection. The efficiency of hESC derivation from human blastocysts by this procedure was about 6%.

Endeavour-1 has been cultured for over one year and has been passaged at least 27 times. It exhibits 10 to 20% spontaneous differentiation after each passage. This line was cryopreserved many times by slow as well by fast freezing methods (Reubinoff et a/., 2001 Hum Reprod 16: 2187- 2194) and recovered after each thawing.

The use of serum-free HFF as a feeder layer and the laser dissection of the ICM provided serum-free and largely xeno-free conditions for the derivation and propagation of this hESC. Example 3. Clonal propagation of Endeavour-1

Approximately 300-400 Endeavour-1 colonies from six well plates were dissected with collagenase type IV (1 mg/ml in PBS without Ca²⁺ 1 ml/well) for 7 minutes at 37⁰C after gently washing twice with PBS. The colonies were allowed to settle at the bottom of 15 ml tube for 5 min and supernatant aspirated. The colonies were dissociated into single cells by using 0.05% trypsin/0.25% EDTA at 37⁰C for 7 min, triturated twice with a pipette. The single cell preparations were re-suspended at 1 x 10⁶ cells/ml in conditioned medium collected from HFF cultured in SR medium for 24 h.

A FACS Calibur (Becton Dickinson) was used to select hESC exclusively as described previously (Thomson JA, et al. Science (1998) 282:1145-1147). Each cell sorted by FACS was dispensed into a well of a 96 well plate containing HFF as a feeder layer and cultured in SR medium in 5% CO2 at 37⁰C for two weeks. The viability of single hESC after FACS sorting was >98% as assessed by fluorescent staining using carboxyfluorescein diacetate (CFDA) to label viable cells and propidium iodide (Pl) for dead cells. Clones obtained were initially passaged by physical dissection in 24 well plates and subsequently into six well plates by trypsin.

Four new clones, E1C1, E1C2, E1C3, E1C4 were obtained after FACS sorting of single cell preparations from Endeavour-1 in 96 well plates (with an overall efficiency of 0.5 to 2%). These clonal lines also exhibited normal undifferentiated hESC morphology, exhibiting colonies with distinct boundaries on culturing.

Example 4. Characterization of Endeavour-1 and Clonal cell lines

The immunohistochemical localization of various stem cell surface markers, including the stage-specific embryonic antigens SSEA-1, SSEA -3, SSEA-4 and the tumor recognition antigens TRA-1-60, TRA-1-81 was carried out using primary antibodies against these surface markers (diluted 1: 40)(Chemicon, VIC, Australia) and visualized by using fluorescein isothiocyanate (FITC)- conjugated appropriate secondary antibodies according to methods recommended by the supplier (Chemicon, VIC, Australia) as described previously (Sidhu and Tuch 2006 Stem Cells and Development 15: 61-69). The pluripotency intracellular marker, alkaline phosphatase (ALP) was assessed immunohistochemically by using a commercially available kit (Sigma-Aldrich, MO USA) following the manufacturer's instructions.

A standard G banding and multicolour spectra karyotyping (SKY) kit was used for karyotype analysis of each cell line according to the manufacturer's instructions (SKY H-10 kit, Applied Spectra Imaging, Inc, Carlsbad, CA). Twenty metaphases for each sample were captured for modal determination. The expression of the pluripotent marker Nanog and differentiation markers for ectoderm (Nestin), mesoderm (Renin), endoderm (α-fetoprotein) and definitive endoderm markers, S0X17, GATA4, and F0Xa2 was assessed by semi-quantitative PCR using Gel Doc System (BIO RAD). Total RNA was extracted using an RNeasy mini kit (Qiagen) with DNase treatment. The first strand of cDNA was synthesized using 5 μg total RNA with MMLV-RT (Gibco) and oligo (dT) primer (Roche). Expression levels were normalized to the expression of a control gene, β-actin.

The ability of the hESC lines to respond to a stimulus for the directed differentiation to definitive endoderm was assessed using a five day treatment with activin A (D'Amour KA, ef a/. 2006 supra). The expression of the definitive endoderm markers SOX17, FOXa2 and GATA was examined by RT-PCR and by immunocytochemistry. hESC from each clone were assessed for the pluripotency in vitro, Colonies were dissected out from wells with collagenase and cultured in SR culture medium in suspension for 3 to 4 days to produce embryoid bodies EBs. The EBs were seeded in gelatin-coated tissue culture dishes and cultured in SR medium without bFGF for two weeks to induce spontaneous differentiation. The expression of lineage markers in hESC cultures after RNA extraction for ectoderm, mesoderm and endoderm were evaluated by immunocytochemistry as described previously (Sidhu and Tuch 2006; supra),

In culture, E1 and its clonal lines, E1C1, E1C2, E1C3, E1C4 formed morphologically normal hESC colonies, with clear boundaries and a compact mass of cells having a high nucleus- cytoplasm ratio. The colonies showed a uniform localization of the pluripotency marker alkaline phosphatase activity. All cell lines also exhibited gene expression for the pluripotency marker Nanog by RT-PCR.

Antibody labelling of E1 colonies demonstrated the strong expression of stem cell surface markers including SSEA-4, TRA-1-60, and TRA-1-81. Some expression of Nestin and α-fetoprotein in otherwise apparently undifferentiated colonies of E1 was also observed. Similar gene expression was observed for the new clonal lines E1C1, E1C2, E1C3 and E1C4. The clonal lines E1C1, E1C2 and E1C4 also exhibited normal karyotype characteristics of the parent line (46 XX). The clonal line E1C3 exhibited reciprocal translocation involving chromosome 15 & 17. All lines showed expression of the stem cell surface markers SSEA4, TRA-1-60, and TRA-1-81.

The pluripotency of E1 and each of the clones E1C1, E1C2, E1C3 and E1C4 was examined under in vitro and in vivo conditions. E1 and each of the clones E1C1, E1C2, E1C3 and E1C4 formed embryoid bodies in suspension cultures. After seeding in gelatin-coated plates E1 and each of the clones E1C1, E1C2, E1C3 and E1C4 spontaneously differentiated into cell lineages derived from all three germ layers, as assessed by the detection by immunohistochemistry of the ectodermal marker, β-lll tubulin, the mesodermal marker, CD34 and the endodermal marker, α- fetoprotein. Cells of the ectoderm lineage (βlll-tubulin positive) were obtained predominately, followed by mesoderm (CD 34 positive) and endoderm (α-fetoprotein) lineages, Subtle differences in the expression of these markers were observed amongst different clonal lines (see Table 2). By utilising these differences, it may be may be possible to more selectively differentiate the hESCs to desired lineages,

The directed differentiation of E1 and its clonal lines by activin A treatment produced endoderm, as revealed by the expression of GAT A4, S0X17 and F0Xa2 genes by RT-PCR. No significant differences in gene expression for F0Xa2, S0X17 and GATA4 after activin A treatment were observed between the cell lines. The implantation of E1 or its clonal lines under the kidney capsule of NOD-SCID mice produced both solid and cystic teratomas. Histological studies using hematoxylin eosin stained paraffin sections of these teratomas revealed the presence of tissues derived from all three germ layers, including gut-like structure (endoderm), cartilage-like structure (mesoderm) and neural ectoderm-like structures (ectoderm). A comparative analysis of various characteristic features of E1 and its clonal lines is shown in Table 1. From this table it can be seen that on implantation E1C2 differentiated into endoderm more than E1 and the other cell lines.

An analysis of the expression of various haematopoietic markers using an immuno- microarray (Medsaic, Sydney Australia) also revealed subtle differences between the parent line E1 and its clonal lines, as illustrated in Table 2, indicating differences which may be used for specific lineage selection.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such modifications and variations.

Table 1. Summary of the characteristic features of hESC clones

Nanog, Nestin, Renin, α-Fetoprotein gene expression determined by RT-PCR. ++ strong; + weak; - absent

Table 2. Summary of phenotypic expression of haematopoietic markers in E1 and its clonal lines, E1C1 , E1C2, E1C3, and E1C4 compared to that in human fetal fibroblasts (HFF passage 1) used as control

Claims

The claims defining the invention are as follows:

1. A human embryonic stem cell (hESC) line or an isolated cell thereof selected from the group consisting of: a hESC line designated Endeavour-1 wherein the hESC line has been deposited pursuant to the Budapest Treaty with the China Centre for Type Culture Collection (CCTCC) on 6

January 2006 under Accession Number C200602, a hESC line designated E1C1, wherein the hESC line has been deposited pursuant to the Budapest Treaty with the China Centre for Type Culture Collection (CCTCC) on 8

August 2006 under Accession Number C200626, a hESC line designated E1C2, wherein the hESC line has been deposited pursuant to the

Budapest Treaty with the China Centre for Type Culture Collection (CCTCC) on 8

August 2006 under Accession Number C200627, and a hESC line designated E1C4, wherein the hESC line has been deposited pursuant to the

Budapest Treaty with the China Centre for Type Culture Collection (CCTCC) on 8

August 2006 under Accession Number C200628.

2. A human embryonic stem cell (hESC) line or an isolated cell thereof which is derived from the human embryonic stem cell (hESC) line or isolated cell thereof according to claim 1.

3. The human embryonic stem cell (hESC) line according to claim 1 , wherein cells of the hESC line display any one or more of the characteristics selected from the group consisting of:

(a) pluripotency; (b) long-term self-renewal;

(c) the expression of any one or both of the stage-specific embryonic antigens (SSEAs) SSEA -3 and SSEA-4;

(d) the expression of any one or both of the tumor recognition antigens (TRAs) TRA-1-60 and TRA-1-81; (e) the expression of OCT-4;

(f) the intracellular expression of alkaline phosphatase (ALP);

(g) the expression of Nanog;

(h) embryoid body formation in suspension culture conditions; and (i) teratoma formation after implantation of cells of the hESC line beneath the kidney capsule of NOD-SCID mice, the teratoma comprising differentiated cells which express characteristics of cells from each of endoderm, mesoderm and ectoderm.

4. A differentiated cell derived from the hESC line or isolated cell thereof according to claim 1 or claim 2.

5. The differentiated cell according to claim 4, wherein the cell exhibits characteristics of a cell from any one of endoderm, mesoderm or ectoderm,

6. The differentiated cell according to claim 5, wherein the cell exhibits characteristics of a vascular cell, a heart cell, a nerve cell, a lung cell, a kidney cell, a liver cell, a spleen cell, an epithelial cell or a pancreatic cell.

7. The differentiated cell according to claim 4, wherein the cell exhibits characteristics of a pancreatic cell.

8. A tissue comprising the differentiated cell according to claim 4.

9. A method for producing a differentiated cell from the hESC line according to claim 1 , wherein said method comprises contacting the hESC cells with one or more differentiation factors, and culturing the cells under conditions suitable to induce differentiation.

10. The method according to claim 9, further comprising co-culturing cells of the hESC line with feeder cells.

11. The method according to claim 10, wherein the feeder cells are human fetal feeder cells.

12. The method according to claim 9, wherein the conditions suitable to induce differentiation are defined serum-free conditions.

13. The method according to claim 9, further comprising screening cells for characteristics of the differentiated cell.

14. The method according to claim 9, further comprising separating substantially the differentiated cells from undifferentiated cells.

15. A differentiated cell produced by the method according to claim 9,

16. A method of treating a disease in a subject, comprising administering to a subject a differentiated cell according to claim 4.

17. The method according to claim 16, wherein the disease is diabetes and the differentiated cell exhibits characteristics of a pancreatic cell.

18. Use of a differentiated cell according to claim 7 in the manufacture of a medicament for the treatment of a disease.

19. A method for establishing and proliferating an hESC line or cells thereof in an undifferentiated form, wherein said method comprises co-culturing the cells with feeder cells in serum free and substantially allogeneic conditions.

20. The method according to claim 17, wherein the feeder cells are human fetal fibroblast feeder cells.