The present invention is directed generally to an avian factor and more particularly an avia factor useful in supporting propagation and/or maintenance of animal embryonic stem cell

The development of methods to produce transgenic animals are an important means of gainin a better understanding of genome organisation and also for improving or introducing desirabl traits in commercial animal production. A number of approaches have been proposed t facilitate gene transfer based on the observation that totipotent cells in the early embryo ar susceptible to manipulation and introduction of foreign DNA. Such approaches have include gene transfer via retroviral vectors, sperm-mediated transfer, PGC transfer and microinjectio

In work leading up to the present invention, the inventors investigated the establishment o embryonic stem (ES) cells as a means of producing transgenic animals. ES cells offe advantages over other methods of producing transgenic animals since they are capable of i vitro genetic manipulation such as targeted mutagenesis by selective inactivation o replacement of endogenous genes and/or the introduction of genes or genetic sequenc encoding new traits.

However, on culture, embryonic stem cells undergo differentiation and lose their stem cel phenotype. Previously this has been overcome by culturing ES cells on a feeder layer o fibroblasts (1).

Primary embryonic mouse fibroblasts and immortalised mouse STO cells have been used a feeder cells in the isolation and maintenance of murine ES cells (2). It has been reported th STO cells have also been used to successfully isolate and maintain porcine ES cells (3 Leukaemia Inhibitory Factor (LIF) has been implicated in the maintenance of ES cells i culture and has subsequently been isolated from mouse (4, 5, 6, 7) and human (8) source

Purified recombinant LIF (rLIF) is also routinely used to maintain mouse ES cells in t absence of feeder cells or conditioned medium (5).

ES cells cultured for a number of generations in the presence of mouse feeder cells or rLI have a reduced capacity to contribute to the germ line (loss of totipotency) when the cultured cells are introduced into an early embryo. There is accordingly a need for facto capable of maintaining ES cells in culture without substantial differentiation.

Accordingly, one aspect of the present invention is directed to a method for maintaining i vitro animal embryonic stem (ES) cells without substantial differentiation said metho comprising culturing said ES cells in the presence of a feeder layer comprising chicke embryonic fibroblasts.

More preferably, the present invention contemplates a method for maintaining in vit embryonic stem (ES) cells from mice, rats, chickens, pigs, sheep, cattle, birds, and other no human animals without substantial differentiation said method comprising culturing said E cells in the presence of a feeder layer comprising chicken embryonic fibroblasts.

In a related aspect of the present invention, there is provided a method for maintaining in vit animal ES cells without substantial differentiation comprising culturing said ES cells und appropriate conditions and in the presence of an effective amount of an avian factor. In preferred aspect of the present invention, the avian factor is derived from chicken embryoni fibroblasts (CEF) or other embryonic tissue such as chick embryo extract (CEE). In a mo preferred aspect of the present invention, the avian factor is a cytokine or cytokine-lik molecule.

The present invention is predicated, at least in part, on the discovery that CEF and CE produce a factor which maintain ES cells in the substantially undifferentiated state. Th present invention extends to the maintenance of all animal ES cells including cells fro

mammals such as humans, livestock animals, companion animals, laboratory test animals an wild animals as well as all avian ES cells including cells from poultry, game birds, cage birds and wild birds. The use of non-chicken ES cells is dependent on CEF or the facto produced therein or in CEE having activity in maintaining the ES cells which could be readil tested by one skilled in the art. In a preferred embodiment, the ES cells are of mouse, rat chicken, pig, sheep or cattle origin.

The invention further extends to the maintenance in culture of human or animal primordia germ cells, haemopoietic stem cells, or cell lineage stem cells, without substantia differentiation utilising CEF or avian factor as herein described, optionally in association wit other cytokine factors, such as steel factor (21).

The CEF of the present invention are a primary cell line with a finite life in culture. The form a confluent monolayer of fibroblastoid cells. The CEF may be prepared from "naturally occurring cells or the cells may first be subject to a range of mutagenic or potentiall mutagenic manipulations such as with chemical mutagenic agents, UN light and geneti manipulations with, for example, viruses, electroporation and microinjection of mutageni genetic material, amongst other procedures. A resulting derivative CEF cell line may produc an altered or derivatised factor having improved or more efficacious properties in maintainin ES cell lines. An additional aspect of this invention extends to such altered or derivatise factors. Derivatised factors where one or more amino acids are deleted, replaced and/o substituted may be produced as described above, or by direct mutagenesis (such as sit directed mutagenesis (22)) of the encoding genomic DΝA or cDΝA followed by polypeptid expression in a suitable host cell. Derivatised factors may also be produced by chemica modification of the polypeptide backbone according to methods well known in the art.

The factor associated with CEF is substantially not secreted or released from the cell a evidenced by the low activity of CEF conditioned medium to maintain ES cells (see Exampl 1). It is probable, therefore, that the factor is cell or cell-matrix associated. The presen

invention, however, extends to a soluble form of the factor prepared by any number o techniques including membrane disruption, cell extraction, physical shearing or othe membrane or cell component solubilising procedures. Conveniently, the factor can be sourced from CEE. Alternatively, the factor may be cloned allowing the production of recombinan factor. Cloning may be by any number of procedures including first purifying the factor, ascertaining N-terminal, C-terminal and/or internal amino acid sequences, deducing oligonucleotide probes from these sequences and then screening a suitable chicken cDNA or genomic library. Alternatively, a chicken cDNA or genomic library could be prepared using a suitable expression vector and expression products screened for their ability to maintain ES cells or by reaction with anti -factor polyclonal or monoclonal antibodies. Suitable cloning methods are described, for example, in Sambrook et al (12) which is incorporated therein by reference.

Another aspect of the present invention provides a chick-derived stem cell factor having one or more of the following properties:

(i) a molecular weight of approximately 15,000-35,000 daltons and preferably 20,000-30,000 daltons as determined by gel filtration chromatography;

(ii) elutes from a 10-90% v/v acetonitrile linear gradient during reverse-phase C ₈ HPLC at approximately 49% acetonitrile; and

(iii) has an amino acid sequence in the N-terminal region of Xaa ¹ Pro Val Ala Gly Tyr Xaa ² (SEQ ID No 6); wherein Xaa ¹ represents four unknown N-terminal amino acids and Xaa ² represents the remaining amino acids of the polypeptide.

Additionally, the chick-derived stem cell factor:

(i) is substantially non-reactive to antibodies directed against recombinant mouse leukaemia inhibitory factor (LIF); and

(ii) is encoded by a gene having a nucleotide sequence in its coding regions of les than approximately 70% homology averaged over the length of the cDN molecule when compared to the cDNA sequence encoding mouse LIF.

Put in alternative terms, the chick-derived stem cell factor comprises a protein having molecular weight as determined by gel filtration chromatography of approximately 15,000 35,000 daltons and preferably 20,000-30,000 daltons, has an amino acid sequence in the N terminal region of Xaa ¹ Pro Nal Ala Gly Tyr Xaa ² (SEQ ID No 6), is derivable from CEF an CEE and is capable of maintaining ES cell in vitro without substantial differentiation in a dos dependant manner. The chick-derived stem cell factor may further be characterized by havin an approximate molecular weight on 12.5% SDS-PAGE of about 50,000 daltons. Th difference in molecular weight between gel filtration chromatography and SDS-PAGE i presumably due to the effects of glycosylation.

Accordingly, another aspect of the present invention provides an isolated chick-derived ES cel factor capable of maintaining in vitro animal ES cells as herein described in the substantiall undifferentiated state. By "isolated" is meant a preparation of the factor as described abov and extends to recombinant and chemically synthetic forms thereof. The isolated factor i preferably biologically pure e.g. 30-80% or greater than 90% purity relative to other chicke derived components as determined by weight, or other convenient means. However, th present invention also extends to conditioned medium or supernatant fluid containing the E cell factor whether produced by recombinant cells over-expressing the factor (for example, b expressing DNA encoding the factor in suitable host cells) or by non-recombinant cells, bu substantially devoid of cells.

Although the chicken ES cell factor has some activity in common with LIF, there does no appear to be any genetic similarity between the two factors, and if any genetic similarit exists, it would be at a level of less than 70% genetic homology. Without wishing to b bound by any mechanism of action, the factor may act through the LIF receptor.

The capacity to maintain ES cells, stem cells and haemopoietic cells in culture allows variou genetic manipulations to be carried out, such as the introduction of foreign DNA for purpos of gene therapy or the production of transgenic animals possessing desirable phenotypes.

The present invention is further described by reference to the following non-limiting Figure and Example.

In the Figures:

Figure 1 is a graphical representation showing the growth of STO cells.

Figure 2 is a photographic representation showing the effect of feeders on mouse ES (D3 clones. A. Inactivated CEF feeders (200x); B. Inactivated STO feeders (200x); C. Gelati (lOOx). Growth of mouse ES colonies after 9 days on

A Inactivated CEF feeders (200 X mag)

B Inactivated STO feeders (200 X mag)

C Gelatin (100 X mag)

Figure 3 is a photographic representation showing the detection of the LIF gene in mouse bu not chicken.

A BRL DNA molecular weight markers

B 90/2 WL genomic DNA digested with BamHI (lOμg)

C 90/35 LD genomic DNA digested with BamHI (lOμg) D 90/3 Australorp DNA digested with BamHI (lOμg)

E Mouse genomic DNA digested with BamHI (lOμg)

F 720bp hLIF fragment (lOOpg)

D PolyA ⁺ RNA from STOs (4.2μg)

E PolyA ⁺ RNA from CEFs (5.1 μg)

F Promega RNA ladder (5μg)

Figure 5 is a schematic representation of the primers used in the PCR amplification of RT

RNA.

Figure 6 is a photographic representation showing a PCR product from STO RT RNA of mouse but absent in chicken. A BRL M _r Markers

B no DNA control

C PCR product from RT RNA from STO cells

D no primer control '

E RT RNA from CEF cells F RT RNA from pBluescript/hLIF

Figure 7a is a graphical representation of a RP HPLC separation profile of chick-embryonic extract with activity in the ES colony assay represented as a bar graph under the corresponding peaks.

Figure 7b is a graphical representation showing a bar graph of the results of a DA- la assay

of pooled fraction IV (\-M) from Figure 7a compared with CEE (t£ ) and TGFβ (BB ).

Figure 8 is a graphical representation of three RP HPLC profiles measured at 220 nm of proteins eluted under different conditions of cation exchange The hatched area under the peaks indicates the regions containing activity in the DA- l a assay. I. 0.1 M NaCl; II. 0.3 M NaCl in. 0.5 M NaCl

Figure 8 is a graphical representation of three RP HPLC profiles measured at 220 nm o proteins eluted under different conditions of cation exchange. The hatched area under th peaks indicates the regions containing activity in the DA-la assay. I. 0.1 M NaCl; ϋ. 0.3 NaCl; III. 0.5 M NaCl.

Figure 9 is a graphical representation of Microbore RP HPLC separated 220 nm profile o fractions 37-38 from 0.1 M NaCl salt step elution. Hatched area represents fraction containing factor activity as seen by the DA-la and ES colony assay.

Figure 10 is a photographic representation showing a 12.5% w/v SDS-PAGE stained with silver nitrate. Lane 1, Fraction 24 Microbore; Lane 2, Fraction 25 Microbore; Lane 3, Sigma Low Molecular Weight Markers; Lane 4, Fraction 23 Microbore; Lane 5, Fraction 26 Microbore.

ABBREVIATIONS

CEE chicken embryo extract

CEF chicken embryonic fibroblast cDNA complementary DNA cES chicken embryonic stem cells C0 ₂ carbon dioxide

DMEM dulbeccos modified eagles medium

DNA dioxyribonucleic acid

FCS foetal calf serum

ICM inner cell mass HC1 hydrochloric acid hLIF human LIF

LIF leukaemia inhibitory factor

M molar

Mr apparent molecular weight

NaCl sodium chloride

NCS newborn calf serum

PBS phosphate buffered saline

PCR polymerase chain reaction PGC primordial germ cell

RT PCR reverse transcribed PCR

SDS PAGE sodium dodecyl sulphate poly aery lamide gel electrophoresis *

TFA trifluoroacetic acid

TGFβ transforming growth factor β TB transcription buffer

U unit

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) thiazolyl blue mLIF mouse LIF rLIF recombinant LIF RP HPLC reverse phase high pressure liquid chromatography

UV ultra violet

EXAMPLE 1

Growth of Primary Chicken Embi onic Fibroblasts (CEF) as 37°C for extended periods CEF's are a primary cell line with a finite life in culture. The primary cell line was derive by trypsinisation of whole tissue from eight to ten day White Leghorn embryos, following th method of Freshney (13). This batch of cells was called CEF. Cells were initially subculture every four to five days by a 1 in 10 dilution into fresh CEF medium (DMEM) supplemente with 10% NCS, 2% chicken serum (CSL), 0.01 U/ml penicillin and 50Ug/ml streptomyci (CSL). Cells were incubated at 37°C, 5% CO ₂ and 95% humidity.

Growth of STO (feeder) cells at 37°C for extended periods

STO cells (12) are thioguanine and ouabain resistant and are a fibroblast line from a SI mouse (13). Cells were subcultured every four to five days from a confluent dish by a 1 i

10 dilution into fresh medium (DMEM) supplemented with 10% v/v NCS or 10% v/v FCS at 37°C, 90% humidity and 5% v/v CO ₂

Mouse Embryonic Stem Cells Mouse ES cells (D3 A) (14) were grown on a confluent layer of mitomycin C (Sigma) inactivated STO cells (15) in ES media, which comprises DMEM supplemented by 5% v/v FCS, 10% v/v NCS and 10 ^" M β-mercaptoethanol (15). Under these conditions, colonies of stem cells formed which contained small cells with a large nucleus and minimal cytoplasm. The nuclei contained one or more dark nucleolar structures. The cells packed tightly together in small nests from which it was difficult to discern the individual component cells. Borders of these colonies were not as discreet as with colonies grown on primary embryonic fibroblasts but tend to mimic the morphology of colonies grown in the presence of LIF only. Plates were split using standard trypsinisation procedure. After several days, small colonies could be discerned.

1. Growth rate of D3 A cells on STO feeders

Crude estimates of growth rates made by determining time for the culture to reach stationary phase after trypsinisation splitting would suggest that under these conditions growth was rapid. Stationary phase was reached with cells within 2-3 days of splitting, to give an initial cell density of 2 x 10 ⁴ cells/cm ² .

2. D3 A morphology on STO feeders

ES cell morphology is maintained when cells are routinely subcultured every 2-3 days and refed as the media is exhausted. If these conditions are not maintained, differentiation occurs within 6-8 days.

3. Identification and characterisation of ES cells

Evidence for pluripotency of mouse ES cells is documented by their capacity to differentiate in vivo into various cell types of all three primary germ layers. Identification of ES cells in

vitro is by differentiation in the absence of feeder cells, normal karyotype and alkalin phosphatase activity (18). Mouse ES cells as well as cells of the ICM are known to sho high levels of alkaline phosphatase activity. This activity declines during progressiv differentiation, resulting in low alkaline phosphatase levels in somatic differentiated cells (9 Feeder cells do not have alkaline phosphatase activity.

4. D3 A morphology on CEF feeders

To determine the effect of CEF feeders of D3 A morphology, the following pilot was set u

A 12 well Costar tissue culture plate was gelatin (porcine skin, Sigma) coated. CEF and ST cells were inactivated and plated at 2 x 10 ⁵ cells/well. A row of inactivated STO cells wer plated as a positive control for normal ES maintenance with a row of wells coated with gelati but without feeders as a negative control for lack of maintenance (see Table 1).

TABLE 1 Arrangement of feeder cells

D3 A cells were trypsinised from a confluent plate grown on STO feeders and plated approximately 10 ⁶ cells/well in the first well for each row. Standard ES media was used a described above. Cells were split 1 in 4 using 0.125% w/v trypsin every 3 days and seede into the next well of the same row. Cells from the latest passage were fed daily with fres ES medium. Cell numbers were counted prior to each split. Representative fields of cell

were photographed prior to each split as a record of moφhology. At the end of the experiment, cells from the final passage were fixed and stained for alkaline phosphatase.

No difference was observed between well 1 in all rows after 3 days growth, however after day 6 in well 2, the cell count on gelatin was a log less than D3 A cells on both CEF and STO wells. Colonies on gelatin were still basically stem cells, however, on the border of each colony, a differentiating cell moφhology was seen. ES colonies on STO and CEF feeders had the same moφhology. By day 9 in well 3, cells on gelatin have a dramatically different moφhology compared with cells from wells 1 and 2. The majority of cells have differentiated into a monolayer of oriented endoderm-like and neuronal-like cells. ES colonies on STO and

CEF still maintain the discreet ES colony moφhology (see Figure 2).

Cells having a high alkaline phosphatase activity stain pink after fixation and treatment with the Sigma alkaline phosphatase assay kit. Cells with low activity do not stain or are stained very poorly.

Strong alkaline phosphatase staining was observed on all ES cell-like colonies grown on STO and CEF feeder layers. Residual colonies of stem cells on gelatin plates stained pink, however, feeder cells and the mass of monolayer cells (see below) did not stain at all.

LIF HOMOLOGY IN CHICKEN

The results obtained above indicated the presence of a factor or factors in CEF's which enables the maintenance of mES cells in culture. To determine whether this factor is homologous to LIF, experiments were conducted to see if a homologous sequence to LIF could be identified in chicken genomic DNA.

Using the hybridisation conditions described by Willson (10), LIF homologies as low as 74% at the nucleotide level (between mouse and sheep) should be detectable. Birds diverged from mammals an estimated 200 million years ago and such a large evolutionary distance would

suggest that bird genes would be less than 70% homologous to mammalian sequences (acros a gene sequence), unless conservation of function has maintained those sequences. functional LIF homology could be present in chickens with only the functional regions in th molecule, such as the receptor binding site, being conserved. If this region is sufficientl large, then it may be possible, using a full length heterologous LIF probe, to detect homology using lower stringency conditions.

To test this, both genomic DNA from chicken blood and polyA ⁺ mRNA from CEF's wer screened. Mouse genomic DNA was isolated from mouse tails and polyA ⁺ mRNA from ST cells was screened for comparison.

1. Southern Blot Analysis

Aliquots of BamHI restriction-enzyme-digested chicken and mouse genomic DNA's (lOμg were electrophoresed in 0.8% w/v agarose gels, blotted onto Hybond N ⁺ membrane an prehybridised and hybridised in 6 x SSC, 5 x Denhardts, 0.5% w/v SDS and 100 μg/m sheared Salmon Sperm DNA. The hybridisation probe (720bp Xho I fragment from pXM.6 which contains the human LIF cDNA (8)) was generated by random labelling using a Bresate Gigaprime Kit, and used at ~10 ⁷ cpm/ml. Filters were washed in 2 x SSC, 0.1% w/v SDS a either 50, 55, 60 and 65°C, prior to autoradiography. At 65°C (Figure 3), a unique 3kb ban was detected in mouse genomic DNA using a full length human LIF cDNA fragment probe

At equivalent loadings, no bands were detected in chicken genomic DNA at this stringency This result would indicate that if a LIF homology was present, it would have to have less tha 70% homology to the human LIF clone. Hybridisation at lower stringencies did not revea hybridisation of a unique band.

2. Northern Blot Analysis

Total RNA from confluent plates of CEF's and STO cell lines was isolated by single ste extraction with an acid guariidinium thiocyanate-phenol-chloroform (16). PolyA ⁺ mRNA wa isolated from total RNA using the Promega Poly A Tract mRNA Isolation System IV (Ca

number 25310). The polyA ⁺ mRNA was concentrated and 5μg RNA/track wa electrophoresed in 1% formaldehyde/agarose, blotted onto Hybond N membrane an prehybridised and hybridised in 50% v/v deionised formamide, 5 x SSC, 1 x PE and 15 μg/ml sheared salmon sperm DNA. The hybridisation probe was made by ³² P labelle transcription of a 720bp Xhol fragment from the human LIF cDNA clone in a pBluescri vector using the T ₃ promoter of the vector. The transcript was made using a Promeg Riboprobe Kit and used at ~10 ⁷ cpm/ml. Filters were washed in 2 x SSC, 0.1% w/v SDS 65°C, prior to autoradiography. Using the hybridisation conditions described above, a -700 b band was detected in STO polyA ⁺ mRNA but no hybridisation was evident for CEF polyA RNA (see Figure 4). This indicates that under these conditions a RNA species of sufficien homology to human LIF is not present in chickens.

3. PCR amplification of Reverse Transcription (RT) RNA

PCR of RT RNA can be used to amplify genes from different species, if regions of sufficien homology can be determined. By comparing the DNA sequence of mouse and human, region of high conservation between the two were selected and complementary oligonucleo tide designed (see Figure 5).

RNA was prepared (11) from confluent plates of COS-1, STO and CEF cells, grown a previously described. RNA transcripts were generated from the T7 polymerase promoter i pBluescript II SKThLIF 720bp Xhol clone to provide a positive control for amplification.

Reverse transcription of the RNA was achieved using 2 μg RNA, 1 x TB, 6U RNasi (Promega), dNTP (Pharmacia) and 10-20 Units of AMV reverse transcriptase. The reactio was incubated at 37°C for 2 hours prior to DNAse treatment, phenol/chloroform extraction an ethanol precipitation. This cDNA provided the template for PCR amplification.

RT RNA was amplified using the primers RILIF, H3 LIF, (Figure 5) Universal and Revers primers (17), at 20pmol/reaction in 0.2mM dNTP (Pharmacia Ultrapure dNTP set Cat#27

2035) Promega 1 x Taq polymerase buffer, 2.5U Taq polymerase (Promega), lOpg template, in a reaction volume of 50 μl. Reactions were overlain with 50 μl Mineral oil and heated to 96°C prior to 25 cycles of 1 minute at 96°C, 20 seconds at 45°C, 1 minute 20 seconds at 72°C in a Thermocycler (Cherlyn Electronics, Cambridge England Cat # IHB 2024). A lOμl aliquot of this reaction was then reamplified using the conditions described above and 10% of the reaction electrophoresed on a 0.8% w/v Agarose/EtBr gel. A 639 bp band was amplified from STO RT RNA after the second round of PCR (see Figure 6). No amplified product was detected from the other samples.

SCREENING FOR CHICKEN ES FACTOR IN CULTURE MEDIUM

Conditioned Medium (CM) was prepared as follows: Corning tissue culture plates (5 x 10cm) were seeded with either STO or CEF cells at 5 x 10 ⁵ cells/plate in 12 ml of DMEM, 10% v/v NCS and 2% v/v CS. After 3 days, the medium was taken off the cells, spun to remove cellular debris, filtered through 0.45μ filters and stored at -20°C.

Costar 12 well tissue culture plates were prepared with and without feeders as described above. Mouse ES cells, D3 A, were seeded at a range of concentrations in different media as shown below. D3 A cells were also plated onto STO and CEF feeders in the presence of the various media to ensure that factors in the CM did not alter growth patterns. Cells were split 3 times over a period of 9 days into each of the media and the microscopic moφhology recorded at Day 7. In the Table 2 below, the increasing number of ES colonies is indicated by an increasing number of "+" signs. A "-" sign indicates the present of differentiated cells.

Table 2 summarises the comparison of the ES colony moφhology after 7 days growth in the different conditioned media. The results show that within 7 days, ES cells differentiate in the absence of feeders or conditioned medium, whereas ES cells grown in the presence of CEF/STO feeders or LIF, are maintained. The proliferation of ES cells on LIF, in the absence of feeders, shows that the ES cells used in these experiments are responsive to LIF and that the conditioned medium does not contain specific inhibitors of their growth. In the absence

of feeders, STO CM supports the growth of ES cells, although a small percentage of cell differentiate. ES cells grown in CEF CM, on the other hand, proliferate but contain a hig percentage of differentiated cells indicating that only a minimal amount of factor is soluble

EXAMPLE 2

PURIFICATION OF CHICK-DERIVED ES CELL FACTOR L SOURCE MATERIAL 1. Materials and Methods

1. Preparation of material for isolation of the chicken factor An alternative source of chicken embryonic material was sought in order to provide a greate source of material from which to purify the chick-derived ES cell factor.

Acid extracted 10 to 12 day chick embryos were tested for activity using the mouse embryoni stem cell lines MBL5 (16), E14 (23) and the DA-la assay (19). The extract was also teste for the presence of TGFβ, which is often present in high amounts in embryonic tissue and i an inhibitor of the DA-la Assay. This can be tested using TGFβ sensitive Mink Lun cells(CCL64).

2. Preparation of Chick Embryonic Extract (CEE)

CEE were prepared from Day 10-12 incubated El 2 chick embryos. Eggs were first rinsed i 70% v/v ethanol. Embryos were then accessed through the shell and immediately the hea was separated from the body. Bodies were washed in phosphate buffered saline (PBS), the roughly macerated and weighed. To each gram of wet weight tissue was added 0.1% v/ trifluoroacetic acid (TFA). The acidified tissue was then blended using a Polytron blender fo

3 minute. The homogenate was aliquoted and stored at -20°C. Upon thawing, the extract wa centrifuged at 8,000φm for 30 minutes at 4°C in a Sorvall RC-5B. The supernatant wa decanted and stored at 4°C.

3. ES Colony Assay

MBL5 or E14 cells were plated 1000/well in Costar 48-well plates. To each well was adde the appropriate dilution of CEE or fractionated CEE to give a range of concentration of CE in IX ES medium (Dulbecco's modified Eagles media (DMEM F-12) (ICN Flow supplemented with 15% w/v foetal calf serum (ICN Flow or Imperial), 0.12% w/v sodiu bicarbonate, 2mM glutamine, O.OlU/ml penicillin and 50ug/ml streptomycin (ICN Flow)). I addition, controlled cultures containing lOuM 2-mercaptoethanol and lOOOU/ml recombinan mouse LIF (rmLIF) were added to the growth medium. Cells were incubated at 37°C, 5 CO ₂ and 95% humidity for 5 days. Growth medium was removed and the cells stained wit Leishmans stain (BDH) or by the alkaline phosphatase assay. ES colonies were score qualitatively as dark staining clumps of cells or as alkaline phosphatase positive colonies respectively.

4. DA-la Assay

DA-la cells are a LIF-dependent cell line derived from mouse leukemia cells. The cells wer maintained in DA-la medium (IX RPMI 1640 medium with 2g/l sodium bicarbonate, withou glutamine (ICN Flow) supplemented with 10% v/v foetal calf serum (ICN Flow or Imperial) 0.12% w/v sodium bicarbonate, 2mM glutamine, O.OlU/ml penicillin and 50ug/m streptomycin (ICN Flow) and lOU/ml of mouse recombinant LIF. Prior to assay, the cell were washed 3 times with 50ml of saline solution, containing 2% v/v FCS without LIF an

centrifuged at l,000φm for 10 minutes. After the final wash, the cells were resuspended i RPMI medium supplemented with 10% v/v FCS (without LIF) to a final concentration o 2X10 ⁵ cells/ml. Assays were carried out using a 96 well flat bottomed plate containing 50u of cell suspension and factor per well. The plates were incubated for three days prior t staining with MTT (see 6 below).

5. TGFβ Assay (20)

Growth of Mink Lung cells (CCL64) is strongly inhibited by TGFβ. Cells were grown a 37°C, 5% CO- and 95% humidity in DMEM-F12 supplemented with 10% v/v FCS, 0.12% w/ sodium bicarbonate, 2mM glutamine, 0.01 U/ml penicillin and 50ug/ml streptomycin. Fo assay, 1000 cells per well in a 96 well flat bottomed plate were added in a volume of 50ul at a concentration of 2X10" cells/ml and allowed to attach for 4 hours prior to the addition o factor. Factor to be assayed was in 50ul volume and a 2X serial dilution (in culture medium) of each sample were made to assess activity. Plates were incubated for 5 days prior to staining with MTT (see 6. below).

6. MTT Staining to measure proliferative cell prowth

An aliquot of lOul of a 5mg/ml solution of MTT (Sigma) dissolved in PBS and filter sterilised was added to each well of a proliferative assay. The MTT solution was then incubated with the cells for 4 hours at 37°C and 5% CO ₂ . The crystals which formed on the proliferating cells were then dissolved in 150ul of isopropanol, 0.04N HC1 containing 1% v/v Triton X-100. Absorbency at 570nm is measured using an Anthos Labtec plate reader. Maximal absorbance is a measure of highly proliferative cells.

7. Alkaline phosphatase staining of embryonic stem cells

A diagnostic feature of the undifferentiated state of ES cells is the positive reaction in the alkaline phosphatase assay. As cells differentiate, their ability to be stained by this method is lost. This enables accurate assessment of the state of ES cells. The method used is

essentially that described in the Sigma procedure number 86 (18) using the Alkalin Phosphatase Leukocyte Assay except that staining was performed in the assay wells. Half th volume of the medium was replaced with Citrate-Acetone-Formaldehyde Solution (CAFS) then removed. Cells were fixed in 100% CAFS for 30 sees. The cells were then rinsed i water prior to the addition of alkaline dye mix. After incubation in the dye mix for maximum of 15 minutes the dye mix is replaced with water for at least 2 minutes to stop th reaction before air drying. *

2. Results CEE showed no significant proliferative effect in the DA-la assay (see Figure 7b). This coul be due to an absence of factor/s which interact with the LIF receptors on the DA-la cells o be a result of other inhibitory factors. Results from the Mink Lung assay indicated that th CEE contained the equivalent of > lOOpg active TGFβ, which is sufficient to inhibit the DA 1 a assay (see Figure 7b).

EXAMPLE 3

PURIFICATION OF CHICK-DERIVED ES CELL FACTOR

IL PROTOCOL FOR PURIFICATION FROM CHICKEN EMBRYONIC EXTRACT

A number of purification procedures were initiated in order to characterise the activity in CEE These included small-scale ion exchange chromatography and RP HPLC to determine th elution profile and the feasibility of maintaining activity of the factor during these steps. Ge filtration of partially purified extract was performed to determine the relative molecular weigh of the factor.

1. Materials and Methods

1. Purification of CEE bv reverse phase (RP) HPLC

The flow chart (Scheme I) below shows a pilot purification of CEE by RP HPLC. On millilitre of clarified CEE was acidified with 0.1% v/v TFA (pH~2.5) and bound to A C-PA column which had been equilibrated in 10 volumes of 0.1% v/v TFA. Protein was eluted i

10 ml 60% acetonitrile (BDH)/0.05% v/v TFA. The percentage of acetonitrile in the eluat was reduced to 20% by the addition of twice the eluate volume of 0.1% v/v TFA. A third o the material was loaded onto a Beckman Ultrapore RPMC C _g (4.6mmX7.5cm) column equilibrated in 20% acetonitrile. A 10 to 90% acetonitrile linear gradient was generated ove 60 minute and 0.5ml fractions collected. Figure 7 represents the profile from the RP HPL gradient (measured at 220nm) with the number of ES colonies supported by the activity o each fraction represented as a bar graph. The activity of pooled fractions from the sam gradient was measured by the DA-la assay.

2. Results

The results of the ES colony assay and the DA-la assay (respectively 7a and 7b) showed tha high activity is detected in the fractions eluting at about 46 to 53%, and more precisely abou 49% acetonitrile. The ES colony assay detected activity in three consecutive fractions at dilution of 1/100. Every fourth consecutive fraction from the RP HPLC was pooled prior to assay by DA-la cells. Activity in the DA-la assay is detected at a dilution as high as 1/100.

This activity is equivalent to 38U/ml mrLIF. No significant activity was detected by eithe assay in any of the other fractions assayed.

These results indicate that the chick-derived ES cell factor remains active in the buffers used for HPLC and that RP HPLC separates this activity from inhibitory factors present in the crude CEE extract. The single peak of activity eluting at -49% Acetonitrile (Pooled Fraction IV) indicates that either the factor is a single component or that a number of factors have identical properties under these RP HPLC conditions.

Scheme I Flow chart for RP HPLC separation

CLARIFIED CEE(IML)

I C-PAK COLUMN (C, ₈ )

ELUTED AT 60% ACETONITRILE

I

CEE DILUTED TO 20% ACETONITRILE

I RP HPLC (C ₈ )

ELUTED AT 10-90% ACETONITRILE i

ASSAY INDIVIDUAL FRACTIONS

BY ES COLONY ASSAY AND

POOLED FRACTIONS BY DA-1A ASSAY

EXAMPLE 4

Molecular weight determination of the chick-derived ES cell factor

Gel filtration of Pooled Fraction IV (Example 3) was performed to determine the appare molecular weight of the factor. This was achieved by gel filtration chromatography at 20 acetonitrile/0.1% v/v TFA at 1 ml/minute with a Beckman Ultraspherogel SEC 2000 colum Standard proteins were chromatographed for calibration. The activity of the fractions after g filtration were measured by the DA-la assay. Fractions containing activity eluted from th column at 40 to 42 minutes. This would correspond to an apparent molecular weight 20,000 to 30,000 daltons under the above conditions and more particularly 25,000 to 27,00 daltons.

EXAMPLE 5 Further purification of chick-derived ES cell factor

Cation exchange Chromatography of CEE

Preliminary purification of CEE by RP HPLC showed a complex of proteins in fraction containing factor activity. As activity is maintained after RP HPLC this provides a suitabl second stage purification. Cation exchange chromatography was chosen as an initial step du to the maintenance of activity of the initial extract at an acid pH.

Two purification schemes were used to separate CEE by cation exchange in a lOOm phosphate buffer pH 6.0 (Scheme II flowchart). The first involved a three step salt elution a 0.1, 0.3 and 0.5M NaCl followed by RP HPLC (Figure 8a). This broad step salt elution wa used to assess the purification potential of cation exchange. A pH of 6.0 in lOOmM phosphat buffer was chosen due to the buffering potential of that molarity phosphate and th corresponding acidity of the extract. A linear salt gradient from 0-0.5M NaCl on Mono resin, followed by RP HPLC, was chosen to assess the actual elution of the protein activity

A salt step gradient was applied to a Pharmacia 1ml SP Sepharose HP column equilibrate in lOOmM Phosphate pH 6.0. One mL of clarified CEE was bound to the column i equilibration buffer. Three column volumes of equilibration buffer was passed over th column. Proteins were eluted by the sequential addition of three volumes of each sal concentration of 0.1, 0.3 and 0.5M NaCl in the equilibration buffer. Prior to loading onto th reverse phase column, fractions were acidified to pH 2.5 by the addition of TFA to a fina concentration of 0.1 %. The fractions were then filtered through a 0.22uM filter to remove an precipitate formed. This material was then bound to a Beckman C _g RP HPLC column in 10 acetonitrile/0.1% v/v TFA. The protein was eluted from the column at 1 ml/minute by a linea increase of acetonitrile of 1.33%/minute over 60 minutes for samples from 0.1 and 0.3M NaC and by a non-linear gradient for 0.5M NaCl. Fractions containing activity in the DA-la assa were then further purified and concentrated by eluting proteins from a Microbore RP HPL using a gradient where the acetonitrile concentration was increased at the rate of 1%/minute.

A summary of this purification is shown in the Scheme II flow chart.

Scheme II Flow chart of purification

CLARIFIED CEE (1ML) I

BOUND TO SP SEPHAROSE IN lOOmM PHOSPHATE pH 6.0

I

ELUTE BY SALT STEP ELUTION 0.1, 0.3 and 0.5M NaCl

I

ACIDIFICATION OF SAMPLE TO 0.1% TFA

I RP HPLC GRADIENT -> DA-la assay i

DA-1A ASSAY POSITIVE FRACTIONS i

MICROBORE RP HPLC STEP GRADIENT -> DA-la assay

->ES colony ^* .say

2. Results

Figure 8 shows the 220 nm absorbance profiles of proteins separated by RP HPLC, fro samples eluted after salt elution at 0.1, 0.3 and 0.5M NaCl, respectively. Factor activity, a measured by the DA-la assay is indicated by the hatched area under the peaks. The profile indicate that the majority of proteins from the CEE elute at 0.3M NaCl. Activity of the facto eluted at 0.3M NaCl at -49% acetonitrile, as expected. Activity was also detected in fraction from proteins eluting at 0.1 and 0.5 M NaCl, indicating that the factor is not eluting from th resin at a single salt concentration.

The profile of total CEE proteins eluting from the RP HPLC step would, however, indicat that differential separation was achieved by the 3 salt steps. The spread of factor activity ove a range of salt concentrations may be indicative of differentially charged glycosylation of th factor. Activity from each salt step which was bound to the RP column, eluted at the sam

percentage acetonitrile. The activity from 0.5M NaCl appears to elute earlier than the other profiles due the fact that a non linear gradient was used to separate this sample. The behaviour of the factor on ion exchange was confirmed by the spread of factor activity found in fractions across a linear (0-0.5M) NaCl gradient eluting CEE from Mono S cation exchange chromatography. Activity was seen to elute continuously from the linear gradient between

0.05M and 0.45M NaCl, with the majority of the activity eluting at between 0.15 and 0.35M NaCl.

Activity eluting at 0.3M NaCl contained too many contaminating proteins for effective elution at this salt concentration despite the presence of high 'factor' activity. Therefore, the factor activity detected in 0.1M and 0.5M NaCl was further purified by Microbore RP HPLC, as these fractions were high in activity.

Figure 9 shows the 220nm absorbance profile of Fractions 37-38 from the 0.1M NaCl RP HPLC elution separated on the Microbore column. The profile would indicate that -9 protein peaks were further separated from these fractions. The hatched area under the peaks represents the fractions containing factor activity as measured by DA-la assay and the ES colony assay. The factor activity was detected by ES colony assay and the DA-la assay as eluting from the microbore column at -49% acetonitrile as seen in the previous RP HPLC elutions.

EXAMPLE 6

SDS-PAGE &Westem blot of CEE using polyclonal antibodies raised against miLIF Apart from LIF, DA-la cells proliferate in the presence of Oncostatin M and IL-3, but are not responsive to CNTF or IL-6. The activity could not be attributed to IL-3 as this molecule has not activity on ES cells. To determine whether the activity found in the fractions containing activity from Figure 10 is LIF related, the samples were separated following 12.5%

w/v SDS PAGE and either silver stained or were blotted against serum from rabbit immunised with rmLIF.

To enable immunoreaction with LIF antisera, proteins were electrophoretically transferred t nitrocellulose(0.45u). The membrane was blocked with 5% w/v BSA in PBS, prior to reactio with a 1/100 dilution of polyclonal antisera raised in rabbits against a Gex-LIF fusion protei (Ab 4855/6). Detection of anti-LIF antibodies was accomplished by a 2 hour incubation o the immunoblot with alkaline phosphatase-conjugated second antibody (Sigma).

Three proteins could be seen at an apparent molecular weight of -55, -50 and ~20KDa after silve staining the gel (Figure 11). The 20KDa band cannot be seen in this gel as it has run off th end of the gel. The ~50KDa protein is the major component of the fraction, representing -8 to 90% of the total protein. It is likely that the 55KDa diffuse band represents differentiall gly cosy lated forms of the 50KDa product. The 20KDa band may represent degradatio products of the major 50KDa protein. By comparison to a known amount of rmLIF, a estimate of 40ng/track for the 50KDa band can be made. It appears as a diffuse band typica of glycosylated proteins. Increasing activity in both the DA-la assay and the ES colony assa corresponds to the increase in concentration of this -50 KDa band.

Reactivity with the LIF polyclonal antisera was not observed in these fractions with > 40n chicken factor. A sample of rm LIF electrophoresed as a control, could be detected at greate than lng.

The purified factor was subject to N-terminal amino acid sequence analysis as a definitiv means of characterising the protein. The first four N-terminal amino acids were masked (du to the presence of glycine). The amino acid sequence of the fifth to ninth N-termina sequence amino acids was determined . This amino acid sequence is as follows:

Xaa ¹ Pro Val Ala Gly Tyr Xaa ² (SEQ ID No 6)

wherein Xaa ¹ represents four unknown N-terminal amino acids, and Xaa ² represents the remainder of the amino acid sequence of the factor.

Table 5 provides a comparison of the N-terminal sequence of the factor with the N-termina sequence of various animal LIF's

TABLE S

Comparison of LIF N-terminal sequence from different species to the N-terminus of the Chicken Factor

1, SEQ ID No 1; 2, SEQ ID No 2; 3, SEQ ID No 3; 4, SEQ ID No 4; 5, SEQ ID No 5; where X represents an unidentified amino acid.

A comparison of this sequence with the sequences in Table 5 clearly show that the factor is not a LIF molecule. A homology search of this peptide with the complete protein sequence of LIF of various species show that no homology could be found, therefore excluding the possibility that the chicken factor sequence is homologous to an internal LIF sequence.

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 variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this

specification, individually or collectively, and any and all combinations of any two or mor of said steps or features.

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2. Evans, M J and M H Kaufman (1981) Nature 292: 154-156.

3. Notarianni, E, S Laurie, S Moor and M J Evans (1990) Reprod. Fert. Suppl. 41_: 51-56.

4. Hilton, D J, N A Nicola and D Metcalf (1988) Proc. Natl. Acad. Sci. USA 85: 5971- 5975.

5. Williams, R L, W Risau, H G Zerwes, H Drexler, A Agussi and E Wagner (1988) Nature 336: 684-687.

6. Smith, A G, J K Heath, D D Donaldson, G G Wong, J Moreau, M Stahl and D Rogers

(1988) Nature 336: 688-690.

7. Tomida, M, Y Yamamoto-Yamaguchi and M Hozumi (1984) J. Biol. Chem. 259: 10978-10982.

8. Moreau, J F, D D Donaldson, F Bennett, J W Giannotti, S C Clark and G G Wong (1988) Nature 336: 690-692.

9. Wobus, A M, H Holzhausen, P Jakel and J Schoneich (1984) EXP. Cell Res. 152: 212- 219.

10. Willson, T A, D Metcalf and N M Gough (1992) Eur. J. Biochem. 204: 21-30.

I I. Gough. N M (1988) Anal. Biochem. 173: 93-95.

12. Sambrook, et al, (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

13. Freshney, R (1987), Culture of Animal Cells: A Manual of Basic Techniques. 117 Allen R Liss, Inc., New York.

14. Doetschman, T C, H Eistetter, J Katz, W Schmidt and R Kemler (1985) J. Embrvol Exp. Moφhol. 87: 27-45.

15. Robertson, E J (1987) Teratocarcinomas and Embryonic Stem Cells: A Practica

Approach. Ed. E.J. Robertson, IRL Press.

16. Pease, et al, Dev. Biol. 141 : 344-352.

17. Promega Biological Products 1990/91 Catalogue, 41.

18. Sigma Diagnostics Procedure #86. Alkaline Phosphatase, Leukocyte Histochemica Semi quantitative Demonstration in Leukocytes.

19. Moreau, et al, (1987), J. Immunol. 138: 3844-3849.

20. Sporn, et al, (1987) J. Cell. Biol. 105: 1039.

21. Godin, I, and Wylie, C C, (1991) Nautre 352: 807-809.

22. Thomas, K R, and Capecchi, M R, (1987), Cell 51: 503-512.

23. Hooper, M, et al, (1987), Nature. 326: 292-295

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: Commonwealth Scientific and Industrial Research Organisation Cancer Research Campaign Technology Limited

(ϋ) TITLE OF INVENTION: A Method for Maintaining Embryonic Stem Cells and Avian Facto useful for same

(iii) NUMBER OF SEQUENCES:

(iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: DAVIES COLLISON CAVE (B) STREET: 10 Barrack Street (C) CITY: Sydney σ» STATE: New South Wales

(E) COUNTRY: Australia (F) ZIP: 2000

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(D) SOFTWARE: Patentln Release #1.0, Version #1.2

(vi) CURRENT APPLICATION

DATA: (A) APPLICATION NUMBER: AU INTERNATIONAL (PCT)

(B) FILING DATE:

(C) CLASSIFICATION:

(vϋ) PRIOR APPLICATION

DATA: (A) APPLICATION NUMBER: PL 3935/92

(B) FILING DATE: 4 August 1992

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: STEARNE, PETER A

(C) REFERENCE/ DOCKET NUMBER: PAS NH

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (02) 262 2611

(B) FACSIMILE: (02) 262 1080

(2) INFORMATION FOR SEQ ID No:l:

(i) SEQUENCE CHARACTERISTICS:

10 amino acids amino acid single linear

(ϋ) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID No:l:

Met Lys Val Leu Ala Ala Gly He Val Pro 1 5 10

(2) INFORMATION FOR SEQ ID No:2:

(i) SEQUENCE CHARACTERISTICS:

10 amino acids amino acid single linear

(ϋ) MOLECULE TYPE: protein

(iϋ) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID No:2:

Met Lys Val Leu Ala Ala Gly He Val Pro 1 5 10

(2) INFORMATION FOR SEQ ID No:3: 0) SEQUENCE CHARACTERISTICS:

10 amino acids amino acid single linear

(ϋ) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID No:3:

Met Lys Val Leu Ala Ala Gly Val Val Pro 1 5 10

(2) INFORMATION FOR SEQ ID No:4:

(i) SEQUENCE CHARACTERISTICS:

(ϋ) (iϋ) (v) ( i) SEQUENCE DESCRIPTION: SEQ ID No:4:

Met Lys He Leu Ala Ala Gly Val Val Pro 1 5 10

(2) INFORMATION FOR SEQ ID No:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-teπninal (xi) SEQUENCE DESCRIPTION: SEQ ID No:5:

Met Lys Val Leu Ala Ala Gly Val Val Pro

1 5 10

(2) INFORMATION FOR SEQ ID No:6: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID No:6:

Xaa Pro Val Ala Gly Tyr Xaa 1 5

(2) INFORMATION FOR SEQ ID No:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID No:7:

CTAGAATTCC CATAATGAAG G 21

(2) INFORMATION FOR SEQ ID No:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID No:8:

GTAAAGGTTA GAAGGCCTGG GCC 23