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WO1994003585A1 - Procede de maintien des cellules souches embryonnaires et facteur aviaire utilise a cet effet - Google Patents

Procede de maintien des cellules souches embryonnaires et facteur aviaire utilise a cet effet Download PDF

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Publication number
WO1994003585A1
WO1994003585A1 PCT/AU1993/000399 AU9300399W WO9403585A1 WO 1994003585 A1 WO1994003585 A1 WO 1994003585A1 AU 9300399 W AU9300399 W AU 9300399W WO 9403585 A1 WO9403585 A1 WO 9403585A1
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WIPO (PCT)
Prior art keywords
cells
derived
factor
chicken
animal
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PCT/AU1993/000399
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English (en)
Inventor
Shanny Leigh Dyer
Philip Jennings
Trevor Lockett
John Kaye Heath
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Commonwealth Scientific And Industrial Research Organisation
Cancer Research Campaign Technology Limited
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Priority to AU45529/93A priority Critical patent/AU4552993A/en
Publication of WO1994003585A1 publication Critical patent/WO1994003585A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C12N15/877—Techniques for producing new mammalian cloned embryos
    • C12N15/8775—Murine embryos
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"

Definitions

  • 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
  • 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
  • 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.
  • LIF Leukaemia Inhibitory Factor
  • rLIF human (8) source Purified recombinant LIF
  • 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.
  • 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.
  • ES i vitro animal embryonic stem
  • 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.
  • ES vit embryonic stem
  • 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.
  • the avian factor is derived from chicken embryoni fibroblasts (CEF) or other embryonic tissue such as chick embryo extract (CEE).
  • 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.
  • 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 D ⁇ A or cD ⁇ A 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 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.
  • the factor can be sourced from CEE.
  • 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.
  • 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:
  • (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.
  • chick-derived stem cell factor is 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.
  • LIF mouse leukaemia inhibitory factor
  • 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.
  • an isolated chick-derived ES cel factor capable of maintaining in vitro animal ES cells as herein described in the substantiall undifferentiated state.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 3 is a photographic representation showing the detection of the LIF gene in mouse bu not chicken.
  • Figure 5 is a schematic representation of the primers used in the PCR amplification of RT
  • Figure 6 is a photographic representation showing a PCR product from STO RT RNA of mouse but absent in chicken.
  • 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
  • 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; ⁇ . 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.
  • FCS foetal calf serum
  • CEF Primary Chicken Embi onic Fibroblasts
  • 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.
  • DMEM fresh CEF medium
  • CSL chicken serum
  • CSL 0.01 U/ml penicillin and 50Ug/ml streptomyci
  • 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
  • DMEM fresh medium
  • Mouse Embryonic Stem 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 ⁇ -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.
  • 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.
  • 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.
  • 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 was 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).
  • 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 mo ⁇ hology. At the end of the experiment, cells from the final passage were fixed and stained for alkaline phosphatase.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • RNA reverse transcriptase Reverse transcriptase was achieved using 2 ⁇ g 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 ⁇ l. Reactions were overlain with 50 ⁇ l 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).
  • CM Conditioned Medium
  • 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 0.45 ⁇ filters and stored at -20°C.
  • Table 2 summarises the comparison of the ES colony mo ⁇ hology 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.
  • STO CM supports the growth of ES cells, although a small percentage of cell differentiate.
  • ES cells grown in CEF CM proliferate but contain a hig percentage of differentiated cells indicating that only a minimal amount of factor is soluble
  • 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 TGF ⁇ , which is often present in high amounts in embryonic tissue and i an inhibitor of the DA-la Assay. This can be tested using TGF ⁇ sensitive Mink Lun cells(CCL64).
  • CEE Chick Embryonic Extract
  • 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)).
  • IX ES medium Dulbecco's modified Eagles media (DMEM F-12)
  • ICN Flow 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
  • DA-la cells are a LIF-dependent cell line derived from mouse leukemia cells.
  • the cells were 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.
  • the cell 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,000 ⁇ m 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).
  • Mink Lung cells (CCL64) is strongly inhibited by TGF ⁇ .
  • 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).
  • 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. *
  • 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 TGF ⁇ , which is sufficient to inhibit the DA 1 a assay (see Figure 7b).
  • the flow chart (Scheme I) below shows a pilot purification of CEE by RP HPLC.
  • CEE 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.
  • FIG. 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.
  • the results of the ES colony assay and the DA-la assay 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.
  • 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.
  • 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.
  • fractions 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.
  • 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
  • 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.
  • 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).
  • 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.
  • the purified factor was subject to N-terminal amino acid sequence analysis as a break 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
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

La présente invention se rapporte à un procédé permettant de maintenir in vitro des cellules souche embryonnaires (ES) animales sans différentiation sensible. Ledit procédé consiste à mettre en culture lesdites cellules souche embryonnaires en présence d'une couche d'alimentation comprenant des fibroblastes embryonnaires de poulets. La présente invention se rapporte également à un facteur de cellule souche embryonnaire isolé dérivé du poussin, pouvant être obtenu à partir des fibroblastes embryonnaires de poulet et d'extraits embryonnaires de poussin.
PCT/AU1993/000399 1992-08-04 1993-08-04 Procede de maintien des cellules souches embryonnaires et facteur aviaire utilise a cet effet WO1994003585A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU45529/93A AU4552993A (en) 1992-08-04 1993-08-04 A method for maintaining embryonic stem cells and avian factor useful for same

Applications Claiming Priority (2)

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AUPL393592 1992-08-04
AUPL3935 1992-08-04

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WO1994003585A1 true WO1994003585A1 (fr) 1994-02-17

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994026884A1 (fr) * 1993-05-14 1994-11-24 Biotechnology Research And Development Corporation Cellules souches embryonnaires destinees a la reproduction d'ongules chimeres et transgeniques
US5656479A (en) * 1992-05-15 1997-08-12 North Carolina State University Avian embryonic stem cells
US5843780A (en) * 1995-01-20 1998-12-01 Wisconsin Alumni Research Foundation Primate embryonic stem cells
EP1149899A1 (fr) * 1994-10-21 2001-10-31 Institut National De La Recherche Agronomique Milieu de culture de cellules embryonnaires totipotentes aviaires
KR100378747B1 (ko) * 1999-02-11 2003-04-07 한미약품공업 주식회사 조류의 다능성 배자생식세포주 및 이의 제조방법
US6759244B2 (en) 2001-11-08 2004-07-06 Art Institute Of New York And New Jersey, Inc. Composite blastocysts (CBs) from aggregates of dissociated cells of non-viable pre-embryos
US8962311B2 (en) 2006-08-09 2015-02-24 Valneva Method of obtaining chicken embryonic stem cells
US9260694B2 (en) 2007-04-24 2016-02-16 Valneva Generation of duck cell lines
US9382513B2 (en) 2002-03-08 2016-07-05 Valneva Method of making an avian cell line
US11230697B2 (en) 2006-09-01 2022-01-25 Therapeutic Human Polyclonals Inc. Enhanced expression of human or humanized immunoglobulin in non-human transgenic animals

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990003439A1 (fr) * 1988-09-22 1990-04-05 Amgen Inc. Procede de transfert de genes dans les poulets et autres especes aviennes
WO1990003432A1 (fr) * 1988-09-21 1990-04-05 Animal Biotechnology Cambridge Limited Derivation de souches de cellules embryonnaires pluripotentielles a partir d'animaux domestiques
WO1991013985A1 (fr) * 1990-03-05 1991-09-19 John Kaye Heath Facteur inhibiteur de la leucemie

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990003432A1 (fr) * 1988-09-21 1990-04-05 Animal Biotechnology Cambridge Limited Derivation de souches de cellules embryonnaires pluripotentielles a partir d'animaux domestiques
WO1990003439A1 (fr) * 1988-09-22 1990-04-05 Amgen Inc. Procede de transfert de genes dans les poulets et autres especes aviennes
WO1991013985A1 (fr) * 1990-03-05 1991-09-19 John Kaye Heath Facteur inhibiteur de la leucemie

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5656479A (en) * 1992-05-15 1997-08-12 North Carolina State University Avian embryonic stem cells
WO1994026884A1 (fr) * 1993-05-14 1994-11-24 Biotechnology Research And Development Corporation Cellules souches embryonnaires destinees a la reproduction d'ongules chimeres et transgeniques
US5942435A (en) * 1993-05-14 1999-08-24 The Board Of Trustees Of The University Of Illinois Transgenic swine compositions and methods
US7071373B1 (en) 1993-05-14 2006-07-04 The Board Of Trustees Of The University Of Illinois Transgenic ungulate compositions and methods
US6998266B2 (en) * 1994-10-21 2006-02-14 Institute National De La Recherche Agronomique Active retinoic acid-free culture medium for avian totipotent embryonic stem cells
EP1149899A1 (fr) * 1994-10-21 2001-10-31 Institut National De La Recherche Agronomique Milieu de culture de cellules embryonnaires totipotentes aviaires
US6500668B2 (en) 1994-10-21 2002-12-31 Jacques Samarut Culture medium for avian embryonic cells
US8273569B2 (en) 1995-01-20 2012-09-25 Wisconsin Alumni Research Foundation Preparation of human embryonic stem cells
US6200806B1 (en) 1995-01-20 2001-03-13 Wisconsin Alumni Research Foundation Primate embryonic stem cells
US7582479B2 (en) 1995-01-20 2009-09-01 Wisconsin Alumni Research Foundation Primate embryonic stem cell line
US5843780A (en) * 1995-01-20 1998-12-01 Wisconsin Alumni Research Foundation Primate embryonic stem cells
KR100378747B1 (ko) * 1999-02-11 2003-04-07 한미약품공업 주식회사 조류의 다능성 배자생식세포주 및 이의 제조방법
US6759244B2 (en) 2001-11-08 2004-07-06 Art Institute Of New York And New Jersey, Inc. Composite blastocysts (CBs) from aggregates of dissociated cells of non-viable pre-embryos
US9382513B2 (en) 2002-03-08 2016-07-05 Valneva Method of making an avian cell line
US8962311B2 (en) 2006-08-09 2015-02-24 Valneva Method of obtaining chicken embryonic stem cells
US11230697B2 (en) 2006-09-01 2022-01-25 Therapeutic Human Polyclonals Inc. Enhanced expression of human or humanized immunoglobulin in non-human transgenic animals
US9260694B2 (en) 2007-04-24 2016-02-16 Valneva Generation of duck cell lines
US9822345B2 (en) 2007-04-24 2017-11-21 Valneva Method of making a virus using duck embryonic derived stem cell lines

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