USE OF DELTA-LIKE PROTEIN TO INHIBIT THE DIFFERENTIATION OF STEM CELLS

Abstract: A method for inhibiting the differentiation of stem cells is disclosed. The differentiation of stem cells is inhibited by contacting the stem cells with a sufficient amount of a delta-like protein (dlk).

Description: USE OF DELTA-LI KE PROTEIN TO INHIBIT THE DIFFERENTIATION OF STEM CELLS FIELD OF THE INVENTION The piesent invention is duected to the use of Delta-like piotem (dlk) to inhibit the late of diffeientiation of stem cells BACKGROUND OF THE INVENTION The diffeientiated cells of some biological systems matui e in stages fiom a common piogemtoi cell usualK called a stem cell Such cells include foi example, hematopoietic. neui al. epithelial, endothelial. and mesodermal cells Stem cells aie able to diffeientiate into matuie cells w ithin one of these systems Diffeientiation may occui thiough uncommitted oi committed piogemtoi cell intermediates The diffeientiation of a stem cell may lesult in one matuie cell Alternatively, a stem cell may diffeientiate into multiple matui e lineages within a biological system, in which case the stem cell is said to be plui ipotent In some cases, the differentiation of a stem cell may lesult in all the matuie lineages of an entπe biological system, in which case the stem cell is said to be totipotent Stem cells aie also able to self-renew The self-i enewal must be delicately balanced by diffei entiation in oider to maintain a healthy level of stem cells Foi example, hematopoietic stem cells constitute appioximately 0 01% of the cells in adult bone marrow These stem cells, which can be recognized by the piesence of the CD34 antigen, aie found in mici o-enui onments associated with stromal cells Hematopoietic stem cells aie induced by various cytokines. such as c-kit and flk-2/flt-3 ligand. to diffeientiate into increasingly lineage-committed progenitois These piogemtoi s differentiate furthei into the \ aπous mature white blood cells, ied blood cells, and platelets of the hematopoietic system Self-renewal of the stem cells requnes then maintenance in an undifferentiated state There are various applications for methods that increase the time duπng which stem cell populations lemain undiffeientiated in vivo and ex vivo Such applications include, foi example, gene theiapy. and concentrating stem cell populations Another application foi maintaining stem cells in an undifferentiated state is cell and tissue transplantation between animals An example of this application, in the case of hematopoietic cells, is bone marrow tiansplantation Attempts to maintain and expand stem cells, especially hematopoietic stem cells, using various cytokines and cytokine mixtuies have been unsuccessful An object of the piesent invention is to piovide methods for the maintenance of stem cells in an undifferentiated state A more specific object is to provide methods for the maintenance of hematopoietic stem cells in an undifferentiated state. Another object is to provide a method for enriching a population of stem cells in a mixture of stem cells and non-stem cells. SUMMARY OF THE INVENTION These, and other, objects as will be apparent to those having ordinary skill in the art have been met by providing a method for inhibiting the differentiation of stem cells, such as hematopoietic stem cells, and a method for enriching a population of stem cells in a mixture of stem cells and non-stem cells. The methods comprise contacting the stem cells, or the mixture of stem cells and non-stem cells, with a sufficient amount of human delta-like protein (dlk). DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for inhibiting the differentiation of stem cells. For the purpose of this application, inhibiting the differentiation of stem cells means pi eventing, or inhibiting the rate of, the differentiation of stem cells. A stem cell refers to any cell that is capable of self lenewal, and of differentiating into committed pi ogenitors of one or more lineages within a group of cells of a biological system Such cells include, for example, hematopoietic, neural, epithelial, endothelial. and mesodeπnal cells Preferably, the stem cells are capable of re-populating at least one lineage, preferably multiple lineages, and more preferably all lineages of a biological system of cells in an ablated mammal The method involves contacting the stem cells in vivo or ex vivo with a sufficient amount of delta-like protein (dlk) The dlk protein and gene is described by Laborda et al in the Journal of Biological Chemistry 268, 3817-3820 ( 1993). Lee et al in Biochimica et Biophysica Acta 1261. 223-232 ( 1995); and Laborda, International PCT Application WO 94/13701 The dlk gene and protein can be isolated and purified as described in the Laboi da et al and Lee et al articles and in the Laborda patent application The dlk gene and protein may onginate from any mammalian source The human gene and protein are prefened The mouse and human dlk amino acid sequences are given in Figures 1 A and I B, lespectively. of the Laborda patent application: the sequence of mouse and human dlk nucleotide sequence is given in Figuies 3 and 2. lespectively. of the Laborda patent application, and the amino acid and nucleotide sequences of human dlk are given in Figure 6 of the Lee et al article These sequences are incorpoiated herein by reference The dlk gene and protein useful in the piesent invention may contain mutations and polymoiphisms in the sequences as described above Preferably, the dlk gene and protein ai e at least about 75% identical, preferably at least about 85%. more preferably at least about 95%. and most preferably at least about 99% identical to the native nucleotide oi amino acid sequence of a mammal, such as of the mouse and human sequences described above As shown in Figure 2 of the Laboida et al. article, dlk is a transmembrane protein that contains sι\ EGF-like l epeats in an N-terminal extracellular domain and a short C-teiminal mtracellulai domain separated by the transmembiane domain Figuie 2 of the Laborda et al article is incorporated heiein by refeience The dlk protein useful in the piesent invention may be a full length protein Alternatively, the dlk may be in a soluble form (sdlk) The soluble protein may comprise the entn e extiacellulai domain, oi at least enough of the extracellular domain foi the molecule to letain its biological activity The extiacellular domain should compnse at least three EGF repeats (foi example, EGF iepeats 1 -3. 2-5, oi 3-6). pi efeiabh at least foui EGF lepeats (foi example. EGF i epeats 1 -4. 2-5. oi 3-6). inoie pi efeiably five EGF i epeats (foi example. EGF i epeats 1-5. oi 2-6). and most pi efei ably all six EGF i epeats The EGF iepeats are shown in Figuie 2 of the Laborda et al article, which is incorporated herein by reference The soluble dlk protein piefeiably lacks both the ti ansmembiane domain and the intiacellulai domain Altematively. the soluble dlk pi otein may contain all or part of the tiansmembi ane domain and either no part of the inti acellulai domain, or as much of the intiacellulai domain that still peπnits the dlk piotein to leta its solubility in watei at l oom tempeiatuie Soluble forms of dlk can be pioduced by methods known in the art Foi example, the soluble dlk piotein may be pi oduced by cloning an appiopπate DNA sequence in a suitable expiession vectoi The DNA is tiansfected into a suitable host cell, such as, foi example. COS. CHO. NIH 3T3. and expressed Optionally, the soluble dlk piotein is expiessed as a fusion protein Examples of fusion protein parrnei s to be expi essed in tandem with the dlk piotein include, but aie not limited to. lgG piotein. metallotheonine. histidine iepeats. alkaline phosphatase and FLAG pi otein b The dlk piotein is then expiessed. and pi efei ably secieted dπectly into the cell media The piotein is hai vested, pi efeiably in sei um-fi ee media, fiom the stable expiessmg cell lines The soluble protein can be purified by methods known in the art A suitable pui ification method is affinity chromotogiaphy The purified protein is used foi any of the utilities descπbed in the specification The piotein can also be used to immunize animals for the pioduction of anti-dlk monoclonal 01 polyclonal antibodies Such antibodies can be used to identify and isolate dlk-contaming cells, such as stiomal cells, that expiess the dlk piotein The sdlk may be used in solution Alternatively, the sdlk may be aggregated by physical means, such as. foi example, heat, pH. etc , oi chemical means, such as chemical cross linking, as is well known in the ait Such "multivalenf forms of sdlk aie bettei able to cioss link the appropriate target molecules on the surface of stem cells, and to pi vide the appi opπate biological signals The sdlk molecules may also be coated on small, inert beads, such as latex beads foi example, oi on the suiface of a containei, such as a cultuie dish If the dlk piotein used in the method of the invention contains the transmembrane legion, the dlk piotein may be attached to the suiface of a cell, or may be independent of cells Some cells, such as some stiomal cells, contam surface dlk naturally An example of such a cell is the sti omal cell line known as AFT024 Alternatively, the cells may not contain suiface dlk naturally, but may be transfected with the dlk gene so as to expiess one oi moie surface dlk pioteins Some examples of cells suitable to be tiansfected with the dlk gene include COS. CHO NIH 3T3. and BFC012 The dlk protein and its vaπants. as desciibed above, pi events, or at least inhibits the late of. the diffei entiation of hematopoietic stem cells Foi this purpose, the dlk protein may be attached to the suiface of a stiomal cell Alternatively, the dlk piotein may be attached to the sui face of a non-stromal cell oi mav be independent of a cell, in w hich case it is pi efeiably used in conjunction with sti omal cells oi othei tactoi s. such as those that exist on stiomal cells, that, in conjunction with dlk. pi event, oi at least inhibit the rate of, the differentiation of hematopoietic stem cells The dlk piotein and its vanants. as desci ibed above, may also be used along with a cocktail of giow th factois The cocktail, including the hematopoietic cocktail desci ibed below, may be teplaced by. oi used in conjunction with. 5-fluoιouι acιl The giowth factoi s aie selected foi inclusion in the cocktail so as to be specific for the type of stem cells that aie being maintained in an undifferentiated state The cocktail has the effect of enhancing the inhibition of differentiation, and of inducing the pi ohfei ation of the stem cells, leading to self-renewal Foi example, a cocktail of gi owth factoi s foi hematopoietic stem cells comprises a mixture of one oi moie of IL- 1. G-CSF. GM-CSF c-kit ligand. IL- 3, IL-6, and flk-2/flt-3 ligand Such cocktails have been desci ibed by Mooie. et al in "Ex Vivo Expansion of CD34 Hematopoietic Pi ogemtoi s in Gi oss, eds , Advances in Bone Maπow Puiging and Piocessing. Pioceeding of the Fourth International Symposium on Bone Manow Puigmg and Piocessing Oilando, Florida. Wiley-Liss ( 1994) The components of the cocktail the 5-fluoιouιacιl. and the dlk may be used at any concentration at which they aie effective The components of the cocktail and the 5-fluoιouιacιl may. foi example, be suιtabl\ used at concentiations of 01-100 ng/ml piefeiably 1-50 ng/ml. and moie piefeiably 5- 20 ngml The dlk may, foi example, be suitably used at concentiations of 1 ng- 100 ugml. piefeiably 01-100 ug/ l mote piefeiabK l-50ug/ml and most piefeiabK 5-20 ug/ml The dlk piotein and its vanants as desciibed above be used on its own, oi in conjunction with a cocktail of giowth factois. such as a cocktail of hematopoietic giowth factois. and/oi 5-fluoιouιacιl to pi event oi at least to inhibit the rate of, differentiation ex vivo The stem cells be found in a mixtuie of stem cells and non-stem cells Foi example amixtuieof hematopoietic stem cells and moie matuie. non-stem cells occuis in peiipheial blood, bone manow oi umbilical coid blood Dunng the inci eased time the stem cells in the mixtuie aie maintained by the dlk in an undifferentiated state, othei. moie matuie, non-stem cells continue to diffeientiate. and ultimately die In this way. the hematopoietic stem cells become emiched and concentiated in the mixtuie of stem cells and non-stem cells Foi example, a mixtuie of hematopoietic stem cells, piefeiably fiom the bone manow, may be removed fiom a patient undei oing chemotherapy oi 0 radiation tieatment Patients w ho will benefit especially aie those being tieated foi cancels, such as those of the white blood cells, 1 e leukemia Foi example, the patient's bone manow is lemoved fi om the patient The bone manow is puiged of any malignant cells, and the patient is subjected to chemotheiapy oi radiation tieatment The chemotheiapy oi ladiation treatment depletes the patient's hematopoietic cells The hematopoietic stem cells ai e maintained e\ vivo in an undiffeientiated state with dlk. as desci ibed e The stem cell population may be expanded at this stage Alternatively, the stems cells may be isolated using eithei negative oi positive selection An example of positive selection is the sepaiation of a stem cell fi action fi om a laigei cell fi action by binding the stems cells to a monoclonal oi polyclonal antibody specifc to a stem cell antigen, such as CD34 Such antibodies may be bound to miciobeads oi to a column matrix in oider to facilitate the isolation of the stem cells An example of negative selection includes passing a cell fiaction thiough a column in which a vanety of antibodies dnected against matuie cell antigens ai e used to bind the moi e matuie cells, allow ing the stem cells lacking these antigens to be sepaiated into a specific fi action The antibodies may be monoclonal oi polyclonal Following tieatment. the stem cells aie leinfused into the patient foi self- l enewal of the patient's hematopoietic system The stem cells aie pi efeiably derived fiom the patient undei going chemotheiapy oi ladiation ti eatment If necessary, the stem cells may be denved fi om a patient othei than the patient undergoing chemotherapy or radiation treatment. The patient is a mammal, preferably a human. Altematively, the stem cells may be removed from a patient and subjected to gene therapy while the cells are maintained in an undifferentiated state by the dlk protein, or a variant, as described above. The presence of the dlk increases the length of time a stem cell is available for gene therapy. In vivo, the dlk. preferably sdlk. may be used to expand the available pool of hematopoietic stem cells. Such treatment is of benefit before chemotherapy or radiotherapy for various malignancies. Increased stem cell populations would allow a more rapid re-population of all blood cells. including leukocytes and platelets, that are typically depleted during such therapies. dlk may be administered to patients by methods know n in the art. Preferably, sdlk is administered by intravenous injection, or is injected directly into the bone manow cavity. EXAMPLES Example 1. Soluble dlk 1. A full-length cDNA clone for human dlk (hdlk) was obtained as described above. A soluble dlk (sdlk) expression construct was prepared by PCR by truncating the cDNA before the first codon of the predicted tiansmembiane domain The positive (5') primer (Primer I ) was located starting eight nucleotides 5' of the ATG codon, and encoded a Hind III restriction site. The negative (3') primer (primer 2) introduced a stop codon and ! 1 a Not 1 site immediately following the codon for the ultimate alanine in the extracellular domain of human dlk. The sequences of these primers are given below. The resulting PCR fragment was cloned into the eukaruotic expression vector pcDNA3 (Invitrogen Inc.). Protein expression w as achieved by introducing the expression plasmid into NIH 3T3 and CHO cells and establishing peπnanent lines of expressing cells. In parallel, constructs directing the expression of sdlk were joined to sequences expressing the coding regions for alkaline phosphatase and for the Fc region of human IgG l . The resulting fusion proteins (sdlk-AP and sDlk-lg) were expressed as indicated above for sdlk. These fusion proteins can be easily purified and used in several types of binding and functional assays Primer 1 : S'-GAG.GGT.ACC.AAG.CTT CCA GAG.ATG.ACC.GCG.ACC.GA (SEQ ID NO 1 ) Primer 2 5'GCA.TCT.AGA.GCG.GCC.GCT.CAG.GCC.TGT.CCC.TCG GTG.AGG.AG (SEQ ID NO 2) Example 2 Cell lines and cultuie The AFT024 and BFC012 stromal celi lines w ere characterized for their ability to support highly enriched fetal liver and adult bone manow stem cells using both in vitro and in vivo assays. Cells were routinely cultured in Dulbecco ' s modified Eagle's medium (DMEM ) containing 10% fetal bovine serum (FBS) and 50 mM b-mercaptoethanol (BME). and maintained at 3 1 °-33" C, 5% CO ; . For long-term cocultures w ith hematopoietic stem cells, confluent monolayers were iiradiated (20 Gy), placed in modified Dexter media (DMEM. 10% FBS. 10% horse serum. 50 mM BME. 0. 1 mM hydrocortisone) and maintained at 37" C. 5% CO : with weekly media changes. NIH 3T3 cells were obtained from ATCC Example 3 dlk expression analysis Total RNAs from stromal cell lines weie poly A selected. Northern blotted, and hybridized to °P-labeled probes according to standard protocols. A 600 bp dlk cDNA clone from the AFT024 subtracted library was used as a probe. cDNA templates foi RT-PCR were prepared using Superscript reverse transcπptase according to manufacturers pi otocols (GibcoBRL) Oligonucleotide primers for the dlk PCR reactions wei e' sense 5'- GACCCAGGCTGCCCC-3' (SEQ ID NO 3 ) and antisense 5'- GGTACTCTTGTTGAG-3' (SEQ ID NO 4) For analysis of dlk expression at the protein level, antisera specific for dlk was generated by immunizing rabbits with a Flag-dlk fusion protein (described below ) Resultant antibodies were purified by affinity chromatogi aphy on Sepharose C1-4B to w hich dlk-Ig (described below) was coupled accordmg to the manufacturers instructions (Phairnacia Biotech Inc.) After elution and neutralization, affinity purified dlk antibodies were dialyzed against phosphate buffered saline ( PBS) and protein concentration was detennined b\ the BCA method (Piei ce) Cell surface expression of dlk in native and tiansfected ( see below) stiomal cell lines was accomplished by flow cytometry with the affinity purified dlk antibody Stromal cell lines in active growth phase weie washed once with PBS/0.5 mM EDTA, and harvested by tπturation in PBS/EDTA Twice washed (PBS/3%FBS) cells were incubated w ith dlk antibody and a similarily prepared iπ'elevant control antibody Specific labeling w as developed by donkey anti- rabbit-FITC (Jackson Immunoi esearch ) Stained cells were analyzed on a Becton Dickinson FACScan usinu Cell Quest softwaie Example 4 dlk fusion piotein pi epaiation The expression plasmid pCD4-Ig contains cDNA for the extiacellulai domain of human CD4 fused to genomic sequences of the human immunoglobin heavy chain This cDNA was cloned into A ' c^RI and Not I sites of pcDNA3 (Invitrogen) to give the plasmid K.B52 3 2 cDNA encoding the extracellular domain of dlk was obtained by RT-PCR with pi imers BP 1 1 and BP 152 using total RN A fi om NIH 3T3 cells as template The primer BP 152 includes the last codon in the dlk exti acellulai domain and contains an f coRl site in fiame with that of K.B52 3 2 The l esultmg PCR fi agment w as cloned into KB52 3 2 via Hind 111 and / coRΪ sites to obtain the soluble dlk-Ig expression plasmid. pdlk-Ig oi pCD4-Ig w ei e tiansfected into NIH 3T3 cells together with pSVNeo and stable clones w ei e isolated aftei selection in 400 mg/ml G4 18 (active wt . GibcoBRL) in DM EM. 10% FBS Pnmei s. sense BP 151 5'GAGGGTACCAAGCTTCGTGGTCCGCAACCAGAΛG-3' (SEQ ID NO 5), anti-sense BP 152 5'-CTC GATCTG ΛATTCGGCCTGTCCCTCGG TGAGGAG-3' (SEQ ID NO 6) The CD4-Ig and dlk-Ig pi oteins wei e hai vested in sei um-fiee media fi om stable expiessing cell lines The soluble fusion pioteins ei e punfied by affinity chiomatogiaphy on HiTiap Pi otein G-sepharose (Pharmacia Biotech Inc ) equilibrated with 0 1 M Tns-HCl, pH 7 6. 0 5 M NaCl and eluted with 0 2 M glycine-HCl, pH 2 8, 0 5 M NaCl Eluted pi otein was dialyzed against PBS Protein concentration was determined by the BCA method ( Pieice) Flag-dlk fusion pi otein w as used to immunize labbits foi the pioduction of dlk antiserum The pi otein expiession plasmid pcDN 3-Flag is a modification of the plasmid pcDNA3 (Invitrogen) and contains 22 base pan s of 5' untranslated sequence, a pi epi otrypsinogen signal sequence, and the coding region for the Flag peptide (DYK.DDDDKI) as well as a B l II lestπction site A cDNA fiagment encoding the exti acellulai domain of dlk w as obtained by RT-PCR using RNA from NIH3T3 cells. The 5' pπmei (BP 155) was designed to introduce an in frame Bgl II site at the 5' end of the predicted mature dlk protein coding sequence. The 3' primer (BP 154) contained λ ' ha I and Not I sites downstream of a stop codon which is immediately adjacent to the last amino acid of the predicted dlk extracellular domain Primers, sense BP 155 5'-GACAAGATCTCAGCTGAATAGCGACCCACCCTGTG-3' (SEQ ID NO 7); antisense BP 154 5'- GCATCTAGAGCGGCCGCTCAGGCCTGTCCCTCGGTGAGGAG-3' (SEQ ID NO 8) The PCR fragment was digested w ith B l II and Not 1 and ligated into pcDNA3-Flag to yield pFlag-dlk pFlag-dlk was ti ansfected into COS cells using DEAE-dextran Affinity purification of the Flag-dlk protein from COS conditioned media was perfonned according to lnanufactuief s directions using the Flag monoclonal antibody, M 1. immobilized on agarose (International Biotechnologies). Example 5 Plasmid constructs and stable ti ansfection Full-length muπne dlk cDNA was obtained by RT-PCR with pπmeis BP 151 (SEQ ID NO 5, see above) and antisense BP 200 5' GCATCTAGAGCGGCCGCGAACGCTGCTTAGATCTCCT-3' (SEQ ID NO 9), using total RNA fiom NIH3T3 cells as template Sequencing confirmed that the resulting product w as identical to the published dlk sequence The product was subcloned into the vectoi pCRII ( Inviti ogen) and then cloned into a retroviral expression vectoi via the pi nnei -encoded Hind l and Notϊ sites Supercoiled plasmid was ti ansfected into BFC0 I 2 stiomal cells by the calcium phosphate method, according to the manufactuief s pi otocol (GibcoBRL) together ith the pZeo ( Inviti ogen) selectable marker and selected in 50 mg/ml Zeocin (Invitrogen). BFC012 cells w ere also ti ansfected with pZeo alone and selected as above. Clones from both selected populations weie isolated and all remaining colonies ( 100-200/dish) were pooled and expanded as populations. Example 6 Hematopoietic stem cells and in vitro hematopoietic assays. Hematopoietic stem cell populations w ere derived from wild type. Ly5.2-C57B1/6J (Jackson Laboratones). day 14 fetal liver, enriched for the AA4 1 . Sea- 1 . c-kit , and lin' 1 " phenotype. by immunopanning and fluorescence-activated cell sorting Adult bone man ow (BM ) was used directly after density centrifugation and immunomagnetic bead depletion or was furthei enriched for Sca-L, c-kit . lin'° " cells by flow cytometry as described Cell sorting and data analysis was accomplished w ith a Becton Dickinson FACS Vantage using Cell Quest Softw ai e Stiomal cell/stem cell cocultures were initiated 12-well trays ith 300- 1000 eni iched stem cells pei well. Cobblestone areas were quantitated by inverted-phase microscopy as described Clonoge c progenitor assays were performed with either fieshly purified stem cells or cells harvested from the stromal cocultures These w ei e cultured in cytokine-containmg semisolid media according to the manufacturers lecommendations (Stem Cell Technologies) Progenitor colonies were scored after 8- 12 days according to established ci itei ia Soluble dlk and control fusion proteins were added to semisolid pi ogemtoi assays at concentrations of 0.1 , 0.5 and 1 0 mg/ml and also to BFC012 stromal cocultuies at concentrations of 0 1 mg/ml Fusion protein was replenished weekly in the st omal cocultures Example 7 Competitive lepopulating ti nsplantation assay Cultuied cells were hai vested, combined with fiesh unfractionated BM obtained fiom congenic C57B1/6 Ly5 1 mice (National Cancel Institute) and transplanted into lethally ii iadiated ( 10 Gy, split dose 3 houi s apart from a 1 >7 Cs source, 1 Gy/min) Ly5. 1 lecipient mice Each mouse leceived 2 X 10 5 competitor BM cells and a fi action of the cocultuied stem cells Mice were bled by capillary puncture of the orbital venous plexus and 100 ml were collected into hepaπn-containing ( 10 U/ml) DMEM. ted blood cells were lemoved by NH^CI lysis The nucleated cells w eie stained foi the Ly5 2 (CD45 2) allelic maikei using eithei FITC-labeled dn ectly conjugated Ly5 2 monoclonal antibody or a biotinylated foim developed with stieptavidin conjugated to Texas Red Cells weie also stained with dnectly conjugated antibodies to lineage maikei s All antibodies and chiomogens weie obtained from Phaπmngen Flow c\ tometnc analysis was done on a Becton Dickinson FACS Vantage using Cell Quest Softwaie SUPPLEMENTAL ENABLEM ENT The invention as claimed is enabled in accoidance w ith the specification and readily available iefeiences and starting matenals Nevertheless, on Maich 3. 1997, Applicants have deposited with the American Type Culture Collection, Rockville, Md . USA ( ATCC) lk-transfected cell line BFC0 12 This deposit was made undei the provisions of the Budapest Treatv on the International Recognition of the Deposit oi ' Mici ooigamsms loi the Purposes of Patent Procedure and the regulations theieundei (Budapest Treat} ) This assures maintenance of a viable cultui e foi thirty years fi om date of deposit The organisms will be made available by ATCC undei the terms of the Budapest Ti eaty, and subject to an agreement between Applicants and ATCC w hich assures uniestricted availability upon issuance of the pertinent U S patent Availability of the deposited strains is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws

Claims: WE CLAIM 1 A method for inhibiting the diffeientiation of stem cells, comprising contacting the stem cells with a sufficient amount of dlk pi otein 2. A method according to Claim 1 wheiein the stem cells aie totipotent 3 A method accoiding to Claim 1 wheiein the stem cells aie plui ipotent 4 A method accoi ding to Claim 1 w hei ein the stem cells aie neui al. epithelial, endothelial. oi mesodeimal stem cells 5 A method according to Claim 1 whei ein the stem cells aie hematopoietic stem cells 6 A method accordmg to Claim 1 whei ein the dlk piotein is human dlk piotein 7 A method accordmg to Claim 1 that takes place ex vι\ o 8 A method accordmg to Claim 7 whei ein the stem cells ai e hematopoietic stem cells 9 A method accoiding to Claim 8 w hei em the hematopoietic stem cells aie in the peπpheial blood, bone manow. oi coi d blood of a human 10 A method according to Claim 1 whei ein the dlk is used in conjunction with a cocktail of growth factoi s 1 1. A method according to Claim 5 wherein the dlk is used in conjunction with a cocktail of growth factors for hematopoietic stem cells. 12. A method according to Claim 1 wherein the dlk is used in conjunction with 5-fluoiOiιracil. 13. A method according to Claim 5 wherein the dlk is used in conjunction with 5-fluorouracil. 14. A method according to Claim 1 wherein the dlk protein is independent of a cell. 15. A method according to Claim 14 wherein the dlk protein is soluble dlk protein. 16. A method according to Claim 1 wherein the dlk protein is attached to the suiface of a cell. 17. A method according to Claim 16 w herein the ceil is a stromal cell. 18. A method according to claim 17 wherein the stromal cell is BFC012 tiansfected with the dlk gene. 19. A method according to claim 17 wherein the stromal cell is AFT024. 20. A method for enriching a population of stem cells in a mixture of stem cells and non-stem cells comprising contacting the mixture w ith a sufficient amount of human dlk protein. 21. A method according to claim 20 wherein the stem cell are hematopoietic stem cells. AMENDED CLAIMS [received by the International Bureau on 22 July 1997 (22.07.97) ; new claim 22 added ; remaining claims unchanged ( 1 page)] 21. A method according to claim 20 wherein the stem cell are hematopoietic stem cells. 22. A method according to claim 21 wherein the dlk is used in conjunction with a cocktail of growth factors for hematopoietic stem cells.

Inventors: LEMISCHKA IHOR R, MOORE KATERI A, PYTOWSKI BRONISLAW, WITTE LARRY, LEMISCHKA, IHOR, R., MOORE, KATERI, A., PYTOWSKI, BRONISLAW, WITTE, LARRY
Applicants: IMCLONE SYSTEMS INC, LEMISCHKA IHOR R, MOORE KATERI A, PYTOWSKI BRONISLAW, UNIV PRINCETON, WITTE LARRY, IMCLONE SYSTEMS INCORPORATED, LEMISCHKA, IHOR, R., MOORE, KATERI, A., PYTOWSKI, BRONISLAW, TRUSTEES OF PRINCETON UNIVERSITY, WITTE, LARRY
Application year: 1997
Main classification: A61K38
IPC codes: A61K38, C07K14, C12N5