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Method Field of the Invention The invehtion relates to a method for identifying a rnodulator of a ce,l signalling pathway; 'ri particular a method for identifying a modulat6r of -a cell signalling pathway that triggers differentiation in cells. Modulators may be id;entified by exposing progenitor cells to candidate modulators. In one 'qspect, the progenitor cells are multipotent or unipotent stetn cells which aro isolated at various stages of differentiatiory, and further cultured together with a potential modulai6r of the differentiation process that acts by promoting or inhibiting. {he eff~:~~ct of cell signalling pathways on differentiation.
Backgtound to the Invention The field of regenerative medicine holds the realistic pramise of reger$eratinq darriaged tissues and organ's in vivo, in patients witF'i co-nditions sucr as cardiovascular disease, neurodegenerative disease, mUsculoskeletal diseaoe, liver disease or diabetes. Techniques for regeneration of dairaged tissue involve either } he repair of existing diseased tissue in vivo using r( ' -.'generative drugs or by 'raplacement of such tissue using cells first prepared in vitro witk or wiihout the use of regenerative drugs and then transplanted in vivo.
In either caSe, the goals of regenerative medicine can only ke realised if specific genes, faotors or modulators controlling cell signalling pathways and the downstream Geliular processes they regulate can be identified. It is thought -that the coritrolled differentia#ioh of stem cells in vitro for example, may provide a source of replacement cells for transplantation. The pluripotency aneF plasticity o,f stem cells allows them to be committed to. a particular cell type following- treatment Jvith certain -coltur.e_ conditiaps. HoWever such an approach relies on the prior identification of factors or modulators that control the cellular and mc?lecular everits of Iiiieage dffferentiation-.
Use of these factors or modulators on stem cells ex vivb could reduce thp lik6iihood of ' spontaneous differentiation of stem cells into divergent lineages upon tP transplantation, as well as reduce the risk of teratoma formation in t~e case of embryonic sterr cells.
Such factors or rnodulators could also form the basis of therapjes that aim td mobilise endogenous stem cells in vivo, or to trigger their differentiation into a celp type that can amplify, repair, restore, replace or otherwise benefit a darraagIed tissue. An example df a factor that affects cell differentiatidn and is Used in thprapy is erythropoietin (EPO). EPO is a naturally occurring protein factor that promc;tes the differentiation of haematopoietic precursors into erythrocytes. RecombinaHt EO
is used to treat anaemia and has a global market of approxiM~tely US$10 bifliori.
In addition to naturally occurring molecules or factors, it is po;I bible to affect differentiation of cells using synthetic modulator-s of bignalling pat1ways, in particralar modulatbrs of pathways controlling differentiation. SB-49711 $ is a small rnolecule crub being developed by GSK that mimics the activity of thrombopoiEI
~in (TPO), a protein factor that promotes growth and production of blodd p+atelets. Tile drug could be used to treat thrombocytopenia: the inability to producQ platelets, which are critically requirqd as components of the clotting process during bjeedipg'. It i's estimated that the market opportunity is approximately US$4-5 billion.
Though regener;ltive drugs such as EPO or SB-497116 act on stem cells, a general method f6r the discovery of regenorative drugs using sterr cells, iri partici~lar embryonic or fpetal stem cells, has not been proposed.
Naive attempts at using stem cells ira drug discovery have irivolved experiMnents in which pluripotent embryonic stem cells, self-renewing adult stem cells or cdli lines have been used in cell-based phenotypic ond pathviray-specific screens of riatural products or synthetic compounds to discover agepts c~fiable of affecting differentiation of these cells (see review by Ding & S.hultz (49-004) Nature Biutechnology 22: 833-840). One of the reasons for using these cells Is trat theV can be easily amplified to yield the quantities required for a screen. Howevdr the approaches in the prior art, in which self-r-enewing, 6ndiffer.ontiat6d stom cells o'r cell lines are used, are found lacking as_drug discovery methodolog-i~s for numkidr of reasons discussed below.
Firstly, the cell types used in the prior art (particularly ES cells and cell jinL-s) may not be physiplogically relevant targets suitable for pharMacological intervention in vivo.
For example, while a factor which is able to cause differentiation of ES
celi~, to e.g.
cardiomyocytes (Wu et al., J Am Chem Soc. 2004:126(6):1590-1) mEly be of use in allowing the creation of cardiomyocytes in vitro from ES cells, the ES cell is not present in the adult and any agent which has activity spqcifically ori the I=5 cell wbuld likely not act as a regenerative medicine in vivo.
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Paretologic data recovery pro. 2 Secondly the cell types used in the prior art (particularly ~S cells and self-reneWing adult steni cells) lie too far upstream of the target oell lineage in the developmental pathway to undergo -directed differentiation to that lineage in resportse to a single applicatiop of a single agent. For instance it is known th~t timed 6pplication of numerous factor cocktails in series are required to differehtiate ES cells into specific lineages, parficularly those which are specified relativ~lly late in development as a resLilt of a relatively complicated process of tissue specification-.
Thii'dly, the cell types used in the prior art (particularly primaty adult stern cells) may be difficult to obtain in sufficient quantities to carry out large soale ~igh-throughpu't drug screening.
kinally, the cell types used ip the prior art (particularly primary edult stem cells) may exl~ibit flighly variable effects in response to drugs, dependirag on the source or the method of isolation and preparation.
W02004/031369 describes methods and cell signalliny pa'thways which permit differentiation of cells - such as stem cells. In the technique desc;ribed in W02004/031369 cells are cultured under multiple culture stepc I -, under a plurality of conditions to modulate cellular pathways and the method prtivides a nieahs of determining the effect of diverse multpple cUlture step regirne~ on celluh'ar propesses such as differeritiation.
Existirng apprbaches to discovering regenerative drugs pre subopt'mal- 'and there exists a need for improved methods to discover such drugs. The prespn~
iriventioti offers solutions to tlxcse problems and provides improved m.ethods for the discovery of drugs - such as regenerative drugs.
Siammary of the Invention The pre'-sent invbntion is based, at least in part, on the fiil ding ~hat qt Is pos'sible to arrest stem cells in a differentiation pathway thus isolating cells of antither type suitable for sdreening. In one embodiment, this is achieved by abtaihing cells of a ffrst type and determining a differentiation protocol which leads to the appearanc{s of a given target phenotype of interest via a progenitor eell, and optionaily further modifying this protocol, typically by varying the cell eulturing media, suph that the
3 differentiatioo process is stalled at a stage in which cells of ano,toer type, preferably progerlitor cells, are present. The identity of th'e cells of the second type eg.
progenitor cells need not be known but their existence in the preparation can be inferred frorn the fact that the phenotype of interest will appear if the original differentiation protocol is followed tb completion, Thbs, in one embodiment, the methods qescribed herein can be used to produce developmental progenitor~ of cell types whose progenitor5 in vivo are not yet known.
One Way of prnducing such cells is to use the method of W02004/031369 in order to discover a series of culture steps leading to differentiaticin and sybsequently modifying or truhcating the prqcess leading to the isolatibn of progepitor cells. The ihventior( recognises that by sequential ekposure to s~,lected agents, cells may kio iso+ated in a partially di4erer'itiated state which is substaritially idehtical to the progenitor pool in vivo, and then screened for factors thpt ipdupe fu'rther differentiation.
The present invention also recognises thbt regenerative drug discovery sc}reening assays alre more likely to identify effective drug candidates if a phy~idlogicaliy relevant progenitor cell is used in a cell based screeri of naturEil pfoducts or r'ynthetic compounds: The physiologically relevant progenitor cells have less delrelopmental ~otential compared to self-renewing embryonic qr adult stem Gells and lie downstream of these multipotent cells in the developmental pathway. F6r example, the target cell type for EPO in the clinical treatment of anoemib is n.ot the e,mbryon'c stem cell, nor the self-renewing adult haematopoietic stem -cell, but rather a more cor-hmitted erythracyte progenitor -cell which lies further alon6 the d~velopmental pathwa~ .and whose developmental potential is far more restricted (Fisher, Exp. B;ol Med 20P3 Jdn;226(1);1-14). If this progenitor population were, availabl~ fo'r use in a drug discovery screening assay, the effect of EPO on regenerating erythroc.ytes would be readily ap.parent. On the other hand, this regenerative effect of 0Q
js not as readily apparent if undifferentiated ES cells are used in a screeh, even though these cellb can be differentiated to erythrocytes using suitable protocols.
Tre~efare physiologically relevant progenitor cell, rather than a r-oultipotent self-repeWing stem cell, shoLj'ld be used in a drug screen to identify regenerative drups.
Ho}nrever an important problehi of using progenitor poriulatigns is that they are Oxtremely difficult to source, they have limited amplification capacity and in some instances they are completely uncharacteriped. For instarlce, certain neural
4 progenitors are well krlown but reside in the living br'ain and 6re thus difficult to source for drug screening assays. Furthermore, the liver is an organ capaple gf rapid regeneration in vitro, and livOr biopsies are relatively easier to source compared to brain biopsies, but the progenitor cell population responsible for livef regeneration has not yet been conclusiveiy identified and therefore cannot be isolated, cultured and used in drug screening assays.
The prosent invention also recognises that physiologically relevant progenitor, cells suitable for regenerative- drug- discovery screening assays cah be derived froni self-renewing embryonic dr adult stem cells, if these cells can be maoe to diff6renti6te up to, but. no further than, the relevant pragenitor stage. There is therpfore a need ip the art for improved techpiques to isolate partially differentO tqd cells in order subsequently to screen for factors that could be used to modulate their d~ff6rer)tiation.
The present invention involves methods of isolating partially i;lifferentiated cell types which comprise physiologically relevant progenitor cells. Ir'i one embodiment, this is achieved Py pbtaining stem cells and subjecting them td the techniquE:s described in in INO2004/031 ~69 to dispover a differentiation profoc6l, and further modifying this Orofocol such that the differentiatioh process is stalled at, or pot progressed past, a stage in which target prpgenitor cells are present. In another embodimPrt, it involve's isolating them from the adult stem cell pool, develop,ino foetus or animal irl various stages df dEvelo.pment dnd optionally modifying them such thot tr'jey can be amplified io vitro. Irl addition, these prclgenitor cells may be used in assays in whic~
natural produc{ s and candidate small molecule modulators of cell signalllng are screened to identify agents which affect cell signalling in the prog0nitor cells, causing, for example, Mobilization, amplification or differentiation, and 'which can then be deveioped into regenerative medicines Aspects and einbodiments of the present invention In a first aspect, there is provided a method for identifying a potential modulafor of a cell signblling pathway, corinprising the steps of: (a) providing a cell of a first cell type, Wherein said first cell type may be differentiated to a second cEll type via progenitor cell by sequentially exposing said first cell type to one or rriorp, preferably two or more reabtion condjtions; (b) adding to or repla6ing at least one of 6aid two or morb reaction conditions to which the progenitor cell has been expo,~,ecl with exposure to orie or more different reaction conditions comprising saiq ppte'rttial modulator; and (c) monitoring the differentiation of the first bell typb to deterrr)ine formation of the second cell type.
In a further aspect, there is provided a method for identifying a potential modulptor of a ceil- signalling pathway, comprising the steps of; (a) providing a cell of p firsi: cell type, whierein said first cell type may be differentiated to a sepond cell type via a progenitor cell by sequentially exposing said first cell type tci twc) or rr!.jore reaction conditions; (b) adding to or replacing at least onle qf said- twci or moFe reaction conditions tb- which the progenitor cell has been exposed- witl'l expospr=e to one or more different- reaction conditions comprising said pc?tential motluiatqr; and (c) monitoriiig the- differentiation of the first cell type to determirje formation of the gecond cell type, wherein differentiation = of the celis to the second cell type is ihdicptive that said potential moduiator modulates the cell differentiation pathway.
The invention also recognises that by derivation from an organi~.h-i in an 4ariy state of development, cells may be isolated in a partially differentiated state and optiorially dmp,Iified, optionally further differentiated, and then screenod for moduiattiTs that induce differentiation to a target cell lineage or phenotype, Accordingly; in one embodiment, the first cell type is obtained pr obtainabie from an embryo or foetus and optionally modified to allow ampiification.
In one errik~e~diment, the progenitor cell is derived ih yitro from a first cell type by oxposure (eg. sequential exposure) to one or more- (eg. tVvo or more) 'reaction condition5.
In anc-th-er embodimept, the cells ara -monitored -for cpll death ini;teadi of cell differentiation. Accor_dingly, the screen may be a toxicity tcreen.
In one embodiment, the reaction conditions corriprise a soreen of ~otential mqduiators (e.g. a screen of at least 100 potential modulators, at least 1p I
Ob potential mocluiators, or ot least 10,000 potential modulators).
In another embbdiment, the first cell type is a self-renewing stei-n cell.
The invention employs cell units. Such units may be singie cells, but are advantal peousiy colonies of two Qr more cells, which are arranged in such a forrn that they are resistant to disruption even during split pool cylturing procedures.
For instance, the cells may be cultured on a solid substrate, such as beads, .as described in more detail below. In the present invention cell units can be isoiated at any stage of the differentiation process triggered by the sequential additibn of gents intp the culture medium. Accordingly, there is provided a meth:od for doterlnining the effect of a pllaraiity of culture conditions on a cell as described above, wherein cell units that are partially differentiated are then isolated and used in the rbethod described for identifying the effect of modulators-on cell signalling pathways.
TypicaNy the cell units used are microc-arriers which are_ small enough- to be transferred to a HTS screehing system in liquid phase and without siibst6ntial disruption of the cell unit. This system greatly facilitates thiE. prc)~uction of quantities c~f differentiated cells for screening, aiid also thO set up of ~he HTS as~~y.
In particular it also allows the use of cells for HTS without any prior disruption of the cells, such as by proteolytic digestion to allow transfer of cells from one vessel to another, which is advantageous since such processds may affect thie immediate differentiation stbtus or downstream differentiation abiijty of cells.
Furthermbre it allows the sutorriated transfer of cells using robotic systetYis.
Thus an impprtant advantage of using cell units - for exampie cell yni~s, grown on microcardiers , to prepare the cellular material prior _to screeriing is that it preserves tr'ie cellular niche that has arisen in the -prep~ration process Eind which tnsy be imiiortant fot fhe downstream differentiation screen. If the celliilat material were prepared in. the. conventiopal way -and disrupted for dispensihg into wblls, theh the nich~ wouid be disrupted. If on th-e other hand the oeiiuiar material were prepare'd for the screen in separate wells, then the resulting- well-td-qvell variation in the preparatihn would make drug 'screening difficult to interpret.
~~1&rantagedusiy, in certain applications thP cell units rrjay be labelled.
Labelling allows the following of the culture conditions to which the cells have been exposed;
thus, any given cell unit can have its label read in o'rder to determine how it has been derived froin the startei r,ell pool or culture. Labellirlg tnay tak4 ~ny Of a~'ariety Qf forms, including nucieic acid labels, radiofrequericy encoded tagip, microsphe~e tags, barcoded tags 6nd spatial encoding of cell units on a surfabe or matrix.
The label may be selected from the group consisting of a virus, an oiigoir~upieotidp, a j)eptide, a fluorescent Gompound, a secondary amine, a haiocarbon, a miicture of stable isotopes, a bar qode, an optical tag, a boad, a quantum cJot and a radiofrequency encoding tag. Two or more labels may everi be selocted ~rom this group and used in combination to label a cell unit, for insfance a beal cprrjpriOing fluorescent corripounds and/or quantum dots. Labelling and specitic la6el~ to be used with cell units are further discussed in our co-pending- appliccj~tion G8015173132-.~
incorporated herein by reference, Celis may be cultured in cell units, each cell unit comprising b'rte or n1ore ceils. in another embodirnent, the cell units are single cells. The cell uni~ may comprise one or more cells adherent to or bounded by a solid substrate. In -a further sr-nbodit'nent, the. solid substrate is a microcarrier or bead. In a still further embodirneirt ihe solid substrate is a Well or medium-permeable barrier. - Ths cialture coniiitiohs may be media to which the cell is exposed. The media may cbiitain one or mbre specific qgents, vVhich influence a cellular process.
In one embodiment, the reaction conditions include any physical or chemickil modium in which cells are isolated and manipulated but suitably the reaction condition is a culture condition to which cells are exposed. Culture conditipns ini:lude growth rnedia, temperature regimes, substrates, atmospheric conditions, phiysical cell handling and the like. Growth rnedia comprise natUral and synthetic ~ubstances that nourish and affect the cells including but not limited to basal media, growth factors, nutrients, buff6rs, chemicals, drugs and the like. The reaction conditions may even conipri$e a screen of potential modulators of a cell signalling pathinray.
The inverition may be used-to monitor differentiation of a first cell type at ariy-stage of development but the inventors recognise that due to their reliative ease of clalture, pluripotencY and therapeutic potential , stem cells maY , be particularlY-suitah~
lo as a first. coll type. Thus, in one- embodiment, the in~ention prbyides a-r-nethnd-a's described above wherein the first cell type is a cell whlcfi has be'en arrested alphg a differentiation pathway between a stem cell and a d14erentiated cell ty0b.
Iri one enibodiment, the -cell type is a primary cell, cell line hr turiiour derived cpll line.
Tthe tissue of origin of the cell type may be selected fram a group cori sisting pf brain, heart, liver, lurig, hair, eye, gut, blood, ear, kidney, Sl~in, toot~, pancreas, rriusc6, bone and vasbulature.
In another embodiment, there is provided for use of a partially differenliated, or progenitor, cell type. The said cell type may be isolated frpm an organism or produce8 from pluripotent cells using a method of deterrnining the effect of cyIture conditions on differentiation; and further subjected to aoethod of identffying the effect of modulotors on cell signalling pathways affecting differe',ntiation.
The methad of idehtifying the effect of modulators on cell signalling pathwayo ~ffectirig differentiation may involve screening potential modulatats usirig the drug ~iscovery teohnique$ commonly employed by pharmaceutical companies (Reinventirjg Drug Discovery, Executive Briefing, Accenture, 1997; -High Perfor'ri-iani;e L)rdg, piscavery;
Executive Briefing, Accenture, 2001).
In another embodiment, there is provided a method for erbosing progenitor cells or partially differentiated cells to a potehtial modulator and then monitorin'g the effect df the modulator on the process of differentiation. In one enlbodiment, the potential modulator is an inhibitor of a cell signalling pathway, In anoiher eiTibodjnient, the potential mbdulator is a promoter of a cell signalling pathway. The effect of a modulator to promote or inhibit cell signalling pathways a,ffecting differentiation, may be assessed by a suitable assay including but not liniited to rtionitoring phenotype, reporter gerie expression, genotype, molecule production, viability, metabolic changes or the proliferative ability of cells.
The invention provides a-method for obtaining progenitor cells a'r partly ddereniiated cells from tissues in developing embryos and foetuses, or inde'eq adults. A's tissues develop thrQuqh the foetal and adult stages, they develop stem celTs whilch are progres'sivPly restricted in developmental capacit,y, ultimately becoming adult stem cells. -Fhus at any point In development of an organism, progenitor cells may be excised from tissues, for instance from the fDetus of an anima( or a:6taman foetus pbtained immediately following an elective abortion. Por irlstance, cells-which comprise the precursors to dopamine-producing cells rnay be isolated from specific regions of the deveioping central nervous system of the foetus. Since cells derived from foetal material are scarce, it may be necessary to ampl'fy thes~ peils.
In this case it Is possible to do this without affecting their differentiation state by trensforming these cells, for instance using an ohicogene 'such as ~'myc.
Soitqbly, the transformation Is reversible and does not lead to differen} iation qf the progenftor cell.
The invention recognises that foetal or adult stenj cell Ir'iateriai may require fU~ther differentiation in order to produce progenitor cells, and this can be achieved by the method disclpsed below fqr other stem cells, such as E,9 cells;
The invention also prnvides a method for obtaining progenitor cells ar partly diffarentiated cells from pluripotent cell lines, includirtg but not limited to lines of embryonic stem cells, by determining a differentiation protocol and perf6rming this in part. In this case the resulting cell population will corriprise one or more pro'genitor cells: it is not necessary to know which proportion of the cells ip the populotlon are progenitor cells, nor to be able to identify these, however their pr~,spnce maly be inferred frorr1 the fact that fully differentiated cells woUld- arisE from these if the said differentiation prptocol is ooncluded (instead of being arrested).
Differentiation protocols typically irivolve subjecting the cells to a t'mp'oralfy specified series of apprppriate culture conditions. Cells giving riso to progenitq'r cE;lls 'may be induced to differentiate alorig a desired developmental pathway usind this n'ietljod of serial cEll culture. Cells may be arrested at any stage of that differentia~ion proa6s, thus obtaining progenitor cells, by interrupting or modifyiog the series of apprcpp'riate culture czonditions. For instance, if a ten day differentiation pr'otocol cpmprisiiig a series of five cell culture steps is reqUired to differentiate ES ce,lls to Knacropt'iages, theh fully performing only three of the steps in this series will resqit in the p~~rtial differentiation of the ES cells along that lineage and wijl allow isolaticjn bf macrophage progenitor cells.
The method described for idehtifying a plurality of cultiare conditicins allows thousands or mitlions of cell cUlture conditions and reagerlts to be tested, in a multiplexed high-throughput assay, to determine the conditions necessary to achieve tlre differentiatipn -of cells.
In another aspect of the invention, there is provided a method for .fdentifying modulators of cell signalling as previously deacribad, wherein a- first _eell is differentiated t'o a second cell type by modulating cell signalfing arrd/or the expression of one or more genes in the cell.
Cell signalling and/or the expression of the genes in the cell can be moqulated by, for example, addition of biomolecules such as factors, growth factors, morphogens, hormones, receptor agonists and antagonists, lipids, antjbodies, drugs and toe like;
or by addition of synthetic drugs, chi;rhicals, mall molecules and the like.
Suitably, the abo've modulators are added in combinations, such as from a cell extract, from a i;,o-culture, in animal serum, or a cocktail prepared in vitro.
Cell signallinp arid/or the expression of a gene can also be modulated by transfeqting gr i?therwise transferrirrg a gene into the cell such thiat it is expre,4sed or over-expressed ln a transient, ligand-induced or permarient mannb'r.
Alte~rnat}1/ely, the expression of thb endogenous gene may be altered, such aa by targeted enhancer ilisertion or the administration of exogenous agents which cause an increase ar ' ~ecrease in expression of the gene. Moreover, the product of the gene may itself be administ.ered to the cell, or its activity eliminated from the cell, to achieye the som6 resUlt. N1ooutetors capable of decreasing the expression cif a gene include interfer'ing RNA or antisense compounds, while modulators capable of decreasing the activity of a proteiii include drugs, antibodies, aptamers and the like.
In one aspect the differentiation of the cell is monitored by obgerving tHe ~ihenotype of the c'ell or detecting the modulation of expression of one or more genes in -qi cell, thereby determining the state of differentiation of said cell. Phenotype determinatiop can be carried but by a variety of techniques, for instance by visual inspe'i:tion of the cell units under a microscope, or using high content se:reening an8 6nalysis instrumQntation (see Cellomics Inc; www.cellomics.com). /,kIternatively differentiation can be detected by observing a marker product characteristic of the differE3ntiateo cell. This may be an endogenous marker such as a particular DINA or aNA
sequence, or a cell protein which can be detected by a ligand, cooversic,m df an enzyme substrate, or antibody that recognises a pbrticUlar Ohbnoiypie marker.
A
diffPreritiation marker may also be exogenous, i.e. one that h~s bpen introduced-into the cell population, for exarimple by transfection or viral transduction.
Examples 6f exogenous markers are the fluorescent proteins- (e.g. GFP) pr cell surface antigens i Vvhich are not normally expressed in a particular cell- Iineage or whi6h ar-e, epjtbpe-modifie-d; or frofn a different species. A trensgene or exogenous rnarker g0ne with associated transcriptional control. elements can be expressed in a manner that reflects a pattern representative of an endogenous gene(s) indicative of phenotype or differentiation state. This can be achieved by associating the gene with a cell type-specific promoter, or by integrating the transgene ihto a particular locus (e.g. soe European patent No. EP 0695351). The markers indicative of differentiati4h may be detected by a variety of techniques, both manual and automated, including observation under a microscope, affinity purification ('ppnning'), br by fluorescenca activated cell sprting (FACS). Accordingly, the proseqt invention provldes a rnethod of moniioripg differentiation wherein the modulation of expreasion of one or more reporter gehes is observed wherein the reporter gene(s) responJ(s) to 6ne or more diffOrentiation states of said cell.
In. a further em4odiment, the expr.esSion of genes involved is monitored on 4 gene chip. GPne nxpression may conveniently be analysed using a gen~, chip pr arrgy tochnoloqy, which is widely avaiiable from suppliers such ~s Affyimetrix.
Adv;antagf;o.usly; the genes employed in this anaiysis ericaoe extracelltilar markers, which may be detected for inatance by immunoassay:
In another embodiment of the invention the differehtiation of a cell is monitored by ~oss of proiiferative ability.
The inventioil- moreover provides methods of culturing stem ceils, and differd-ntiated cells detived from stem cells in vitro, adherent to microcarriers, sueh as beads.
iVlicrocarrier culture has significant advantages, including tha scale=up of cultures, and also allows units of stem cells to be exposed tq selected culture conditions as required in order to obtain the desired growth and/or differentiation coHditiort .
The potential modulator may comprise an organic or ingrganic small molecule, a r{atural or derlvatised carbohydrate, protein, polypeptide, Oeptide, giycoprofein, nucleic acid, DNA, RNA, oligonucleotide or protein-riucleic adid (PNA). In Fnother embodiment, the potential modulator is obtained fror'i d IPbrary -of small moiecules with drug like prbperties.
In _a_ -further aspect, there is- provided a modulator of a cell signqiling pathyvay obtained or obtairrable by the methods described herein.
In another aspect there is provided a pharmaceutical composition com~irising the modulator together with a pharmaceutically acceptablp carrier; diiuent or expient, In a further aspect, there ig prpvided a partially differentiated coll, whic~
has been differentiated in vitro frorn a stern cell and arrested alorig a tlifferentietion pathway between a stem cell and a differentiated cell type. The partially differeqtiated cell may be a neuronal or haematopoietic cell. The pa{tlally differpntiated cell may be a bipotent cell. The partially differentiated cell may be a unipotent cell.
In a further aspect, there is proyided a method for identifying a modulator of a cell signalliny pathway (eg. a regenerative drug) comprising the use of a progenitor cell.
,. ~
In a further aspect, there is provided a method for identifying a rnodulator of 4 cell s1gn6Iling pathway (eg. a regenerative drug)- comprising the luse of a partially differentiated cell, which has been differentiated in vitro from a ste'rn cell and arr~sted along a differentiation pathway between a stem cell and a differeritiated cell type In a further aspect, there is provided i:he use of a progenitdr eell iii a drhg scrgening assay to qdentify-a modulator of a cell signalling pathw~y (eg=. a regene:rative drug).
In a further aspect, there is provided. the use of a partially differentiated ce,ll, wtiich has been differentiated in vitro from a stem cell and errPSted ~Iong a qifferenliafiioii pathway bet~nreen a stem ccll and a differentiated cell typ;e in a dru7g scrdenir9g assay to identify a rhodulator of a cell signalling pathway (eg. a rc6enero'tjve drug).
In a further a~pect, there is provided a method for differentiating bn ernbryonic atem cell into a progenitor of the myeloid lineage, comprising the use of a gelatin rnicrocari'ier (eg. a CultiSpher microcarrier).
In a further aspect, there is provided the use of a gelatin microcarri,e,r (eg. a CultiSpher microcarrier) for differentiating embryonic 4tem cells into haematopoietic progenitors.
In a further asp>/ct, there is provided a method: for producing a haematopoietic cell ftom a siem cell in vitro comprising exposing said gtem cell to oni: or rnqro, preferably, tVvo or more, readtion conditions, wher-eiri sa;F; rnaction copditions comprise incubating said stem celLwith: (a) retinoic acid, dimethylsulphoxide (DMSO) andfor stem dell factor (SCF); and (b) insulin, stem cell factpr ($CF), TGF
b'~ta 1, PMP2, F3MP4 and/or TPO; and (c) IL-3, IL-6, TPO, EPO arid/or M-CSF.
In one embodiment, the steni cell is seeded on a microcarrier - soch a,5 adelatin microcarrier.
In one embodinient, the stem cell is contained in an IMDM basal mediurn or a Streamlirne Haematopoietic Expansion Medium.
Iii one embodiment, in step (b) insulin alone is used.
In one embodiment, in step (b) SCF, TGF beta 1, 13M1'2 and TPO is used.
Ih one embodiment, in step (c) IL-3 and. IL-6 are used. I-n another.embodimont, TPO, EPO and/or M-CSF are alsp used.
In ane embod)ment, the step (e) is performed on day 1.
In dpe empod-iment, the step (b) is performed on i1ay 4, In one embodiment, the step (c)- is performed on day 6.
Detailed De.4crlption of the Invention De-finitions Culture Cqnditions As used hprein, the term 'clulture r;onditions' refers to the envirotimegt which c+ells are placed in or are exposed to in ordq to jW+dmote growth or differentiation of said cells. Thus, the term refe'rs to thp mediu~n, tem0raturP, atmospheric conditions, substrate, stirring condition:r and the like which m4 affect the grouvth and/ or difFerentia#ion of cells. More particularly, the term refers t specific agpnts whi-oh may be incor.por-eted into culture media and woich may iiifluence the growth arin/or differentiation of Gells.
Cell A cell, as referred- to herein, is defined as the s,rriallest structural urilt of an organism that is capable of independent functioning, -or a_sin_gle-celled etganism, consistinp of o,ne or more nuclei, cytoplasm, and various orgarielles, 411 surrounded by a sernipermeable cell membrane or cell wall. The cell inay be prokaryotic, eukaryotip or archaebacterial. For example, the cell may be a eukaryotip cell.
Mammalian cells are suitable, especially human cells, Cell~ may be{ natiiral or modified, such as by genetic manipulation or passaging ;n culture, to achi'eve desired properties. A stem cell is defined in more detail bolow, and is a totinotent, pluripoteht or multipotent cell capable of giving rise to mtire ttialn one differentiat~d cell type. Stem cells may be differentiated in vitro to gi'v, e rise tb differdntiateEl cells, which may themselves be multipotent, or may be terminWly differentiated. Ceils differentiated in vitro are cells which have been created artificially by exposing stem cells to one or rnore agents which promote cell differentiatipn.
First cell type In one embodiment, the first cell type is a cell that retains the 6bility to renew itself thrwgh cell division and can differentiete intr!3 a wiFie range of specialized cell types. In another embodiment, the first cell type is a cell that i$ less differentiated than a progenitor cell. In another embodirnent, the first cell type is a stem cell, as described hereirT helow, The stem ceil may be, for exanrple, an embryonic stem cell or an adult stem cell. The cells of the first lype n-iay be-differentiated intb a certain lineage of a second cell type before the cells of the seeond cell type are screened. Thus, in ortie embodiment, the firsf celi type is differentiated to a second cell type by exposing (eg. sequentially exposin,q) the first cell type to tWo or more (eg. three or more, four or more, or five or mqre) reaction conditions.
$ebond cell iLype In one embodiment, the second cell type is coll that ct ' in only differentiate, but it cannot renew itself anymore. The second cell type rpay be more limlted in the kinds of cells it can become than the first cell type. The second cell may be mor~ differentiated than the first cell type. The seGond ceil may kie riidre differentiated than the progenitor cell. In one embodiment, the second eell tyNe is a partialiy tlifferentiated cell, which ha$ been differentiat~-'d in vitro froi-n the first cell and arrested along a differentiation pathway betweerr a ceill of the ~irst type (eg.
a stem cell) and a differerltiated cell type. In another embodiment, the se5aond i';ell typp is a ceil with a targ'et cell phen,otype.
Regeneirative medicEne or ;drug The term 'regenerative drOg' refers to a natural dr synthetic ubstAnce which_ acts on a- stem cell or progenitor cell and is thNs able to regenerate or repair a tissue or organ of the body. 'Regent~ative medicine' refers to the samo, or to the disciplirie of regenerating tissues or organs es 0 melical freatmept, Regenerative medicine encompasses cell replacement therapies, and/or the administratlon of regenerative drugs to patients.
Progenitor A$rogenitor' or 'progenitor cell' is a cell type which lies upstrdam of a mote differentiated cell, but downstream of a true stem cdil. Progenitars are not typically capable of long term self-renewal as are true stern cells, ahd their developniental potential is more limited than is that of stem cells. For inslanc,e CFU-E
and BFU-E are erythrocyte-committed progenitor dell popuiatipns, wherc;as the LT-HSC is a self=renewing and multipotent haematopoietic stem cell and the ES
cell is a self-renewing arid plurii'iotent stem cell. Differentiated eryth'rocytes can be defived from CFU-E which in turri can be derived from LT-HSO which in turn cg'n fye derived from ES cells.
Cell signalling The term 'cell signalling' refers to the molecular mechanisms whereby cells detect and respond to external stimuli end send messages to other cells. Cell signalling therefore includes -transcriptional and translational -contrqls and.
mechanisms as well as signaftransduction mechanisms.
Modulator The term 'rnodulator' refers to any factor that can vary the state of a cell, changing it frorn one state to another. In the contbxt of the invention tfiis refers to modulation of cell signalling processes. Modulator's may irihibi$ or promote.parkicular cell signalling pathways. They may take the form of ngtur-al products or chemically synthesised rriolecules; for example, an organic or inorganic small molecule, a riatural or derivatised carbohydrate, protein, polypeptide, peptide, glycoprotein, nucleic acid, biVA, RNA, oligoriiacleotide or protein-nucleic acid (PNA).
Mqdulators also incldde agonists or antagonists. Modulators that are inhibitors include but are iiot limited to: npnspecific, irreversible, reversible - competitivQ and nbricompetitiVe inhibitors. Modulators that promote ceil signalling stjmulate or enhance the effect of a partic;,ular mplecular pathway on the cell and incluoe but are hdt limited tip: agonist:;, agonist mimetics, co-factors, Promotp-rs and the like.
Amptificatiod ampl ificatio-n' refers to a process by which an increase in magnitude or numb6-r of cells, cellular components or cellular -processd's uccurs: In particular amplification refers to a process of increasing- cell nptnbers in a cell culture system.
This may occur by increasing the rate of proliferatiop -or survival I of cells in tlte-.~ystem.
C6impouhl The term 'compound' is used herein irt arcdrd'artce with the m6aning nbrmaily a~,signed thereto in the art. The term compound is iased irt its bi-oadest sense i.e. a substance comprising two or more elements in fi%ced proplortions, inclUdirtg molecules and supramolecular complexes. This definition includes small molecules (typically <500 Daltons) which make up the majority of pharmaceuticals.
Howev6r, the definition also includes larger molecules, including polymers, for example polypeptides, nucleic acids and carbohydrates, and supraniolecular complexes thereof.
High-throughput screening The term 'high- throughput :i~reonin,y rr:fers td the large-scale, trial-and-error evaluation of compounds in a parallel tai-get-based or ~.ell-based assay.
C,ompound library A'compound libtary' is a group bf diverse cQmpoynds that caii be useo to identify new lead candidates in the drug dt'scovery process.
Compound librEiries may be generated by any means known in the art, inclGding cj6mb'inatorial chemistry, compound evolution, or purchased from commercial sourcea ;such as Sigma Aldrich, Discovery Partnars International, Maybrid'ge and Tripos. A
repertoire advantageously comprises at least 102, 103, 104, 105, 106, 107, 1 O8, 1091 1010, 10' or more diff-erent compounds, which may be related or Unrelated in structure or function.
Modulatiori The term modulation is used to signify an increase and/or d.errea,ge in the parameter being modulated. Thus, modulation of gene exprossion includes both increasing gene expresiion and decreasing gene expressiori.
Cellular prpcess A cellular process is any characteristic, function, process, event, cause or effect, intracellular or extracellular, which occurs or is ob5erved or which can be attributed to a c6ll. Examples of cellular processes include, but are not limited to, viability, senescence, death, pluripotency, mdrphblogy, signalling, binging, recognition, molecule production or destruction (degradation), .mutbtion, pro{ein -folding, transcription, translation, catalysis, synaptic tr.ansmissio.n+
vesicular transport, organalie function, cell cycle, metabolism, proliferation, division, dif#erentiation, phertotype, gehotype, gepe expression, or the control of these prpcesses.
Celi- unit A group of cells, which may be a. group of orie. Pools of c6ii units rnay be sorted, stibdivided and handled without substantially dissociating the- cell urvits the'rriselves, sucf~ that the cell unit behaves as a colony of cells and each cell in the cell unit ip exposed to the same culture conditions. For expinple, aoell unit may corhprise a bead to which Is adhered a group of cells.
T'otipoteht A totipotent cell is a cell with the potential to ~ifterentiate into any type of somatic or germ cell found in the organism. Thus, any desired cell my b~
derived, ~, i py ;ome ~neahs, from a tbtipoterlt cell.
Pluripotept or Multipotent A pluripotent or multipotent cell Is a cell which may differentiate into more than one, but probably not all, cell types.
Label A label or tag, as used herein, is a means to idenl;lf~ a Gell unit dpd/or d-etermine -a culture condition, or a sequence of culture conditions, tci which the cell unit has been exposed. Thus, a label may be a group of 17bels, eabh a4ed at a specific cultiaring step; or a label added at the beginning or the experinjerit 1nd,hiek is modified according to, or tr-acked d'uring, the culturing steps to which the cell unit is exposed; or simply a.positional reference, which allows the culturihg steps used tb bb-deduced. A label or tag may also be a device that reports pr reqords the location or tht identity af a cell unit at any one time, or assigns a unique idehtifier to the ceil unit.
Examples of labqls or te9s are molecuies of unique sequence, structure or ma~s; or fluorescent rrlolecuies or objects such as beads; or rbdiofr6que~cy and other transponders; or objects with unique markings or coiours or shOpes.
Exposure to culture conditions A cell is exposed tci culture conditions when It is placed in contact with a medium, or grown under conditions which affeiA qne or more celluiar process(es) such as the growth, differentiation, or metabolic state of the cell.
Thus, if the culture conditions comprise culturing the ceii in a medium, thE.,ceil is placed in the medium for a sufficient period of time for it to have an effect.
LikIL-}nrise, if the conditions are temperature conditions, the cells are culiured at the desired temperature.
Pooiing The pooling of one or more groups of cell. units invoives the admixture of the groups to create a single group or pooi- which comprises- cell -units of rnore than -one background~ that is, that have been exposed to mar than otie different sets of culture-cond. itioqs. A pool may be subdivided further irito grouos, eithqr -randomly or non-rarldomiy; such groups are not thernseives 'poois' for the present purposas, but rn6y thomseives be pooled by combination, for example after expospre to differerlt bets of culture Cdnditions.
Proliferatipn Cell growth and bell proliferation are Used interchangeabiy herein to denote multiplication of cell numbers without differentiation into dlfferent cell tylies or lineages. In other words, the terms denote increase df viable cell numbers. In one embodiment, proiiferatiori is not accompanied by appreciabW changes in phenotype or genotype.
Differerrtl'ation Cell differentiation is the development, from a cell type, lp a different cell type. For example, a bipotent, pluripotent or totipoteht cell may differentiate into a neural. cell. Differentiation may be accompanied by proliferation, or may be independent thereof. The term 'differentiation' generally refers to the acquisition of a phenotype of a mature cell type from a less developmentally d0fjned cell ty0e, e,g. a rieuron, or a lymphocyte, but does not preclude transd'ifferentiation, whereby one mature cell type may convert to another mature cell type O,g. a neurbn to a iymphocyte. lh- this application 'differentiation.' will be taken to mean 'de=
differentiation' ond vice versa. Commonly, 'de-differentiatidn' refers to the acqUisition -of a phenotype of a less mature cell type from a more deveioprnentally Oefidied cell type, e.g. a myocyte becoming a myogenic precursor. IOe-differen)iation may be followed -by further differentiation to revert to the original (~qll iype, or to a furi.he'r ce)l type [Ding & Shultz (2004) Nature Biotechnology 22: 63-840, alnd refer,onces therein].
Differenti:ation state Tae differentiation state of a pell Is the level to which a cell has differentiated along a particular pathway or lineage.
State cif a cellular process The state of a cellular process rPfers fo Wvhether a cellular prodess is occurring or not and in complex cellular processes can ilenote a particular step or stage in that cellular process. For example,. b cnllular differentiation pathway in a cell may 'be inactive or may have been induced and may comprise a number of discrete steps or components such as signalling eVents characterised-by the presence of a characteristic set of enzymes or iritermediates.
Gene A gene is-a nucleic acid which encodes a gene product, -be it-a pbly~eptide or an RNA gene product. As used herein, a gene in-cludes at least tle cod~ng sequence Which encodes the gene product; it may, optiorxally, include one or rnore recjulatopy regions ne~sessaryfbrthe transcription and/or translation of the coding.
seqUence.
Gene F?roduct A gene product is typically a protein encpdej by a gene in the conventional manner. However, the term also encompasses non-polypeptide gene products, such as ribonucleic acids, which are encdded by the gene.
Nucleic apiit synthesis Nucleic acids may be synthesisad according lo apy available technique. In one embodiment, nucleiq 'acid synthesis is automated.
Moreover, nucleic acids may be produced bji biological replication, sucij as by cloning apd f'eplication in bacteHel or eukaryotic cells, according to pr'ocodure's known in the art.
Qifferential Expressiori Genes Which are expressed at different lev~els iri response to cell dulturd'conditions can be identified by gene expressjon analysis, such as on a gene ar~ay, by methods known in the art. Genes whiph ar,e diffe'rentially oxpressed dis~lay a greater or lesser quantity of mRNA or gene product in the cell under the test conditions than under alternative conditio-ns, r-elative-to overall gerje expression levels.
Transfection Genes may be transfected into cells by ;Iny appropriate mpE I
jtns. The terrh is Used herein to signify conventional transfectiort; for -exarnple using calcium phosphate, but also to include other techniques for transferring nucleic ac'ds into a c,~ell, inclUding transformation, viral trarisduction, electroporation and the likte.
Cell-based assays Cell-based assays are an important part of modern oiornedical s'ciences and comprise any assay that involves a step in which a cell is used. Cell-based 6ss~ays can be used across nearly all stages of the pharhiaceutical drug discovery ano development process, and are valuable in providing information about how a compound is likely to ihteract in a biological systeFn, not just ~bout how it intbr=acts-with a potential drug target in isolatiQri.
For example, cell-based assays can be used to- identify and ~alidate poteritial -drub targets. Cell-ba'sed assays have been developed that cqn-be used to i'dentify genes or cellular pathways involved in -disease processes, to determine the functions of-target genes, or to measure phenotypic chan-ges that may be induced upon a-ctivation of certain genes or their products.
Cell-based assays can also be used in drug discovery For lead-eompdund Oiscovery, selection, and optimization. l)nlike the biochemical assays that are often used in traditional high-throughput-screening assays, cell-besed assays can provide information relating to drug properties such as absorption, permeability, selectivity, specifipity, anc9 metabolism. As a result, lead compoulnds that are selected after cell-based screening are better aharacterized, more likely to provide, valuable leads and less likely to be eliminated in subsequent phases of the drug discovery process.
A major applitation of cell-based assays is in toxicity screenin,. A crucial iiart of drug discovery~ and development is the screening of druii candidates to elimihate csompourrds tkat will cause side effects. However, curront methodoloo_ips_ are-largely irradequate, 'qnd in particular the use of animal rnodels foi= toxicity screening is expPnsiva and time-consurriing. In addition, animol models can be Linrelidble, peoause results in these models do not always accurately prpdict how a compound will perform in humans. Th.us human cei1-based screening is-suitak-le.
Scr-aening assays and HTS
Hiqh thr6ughput screens (HTS) can be used in the present 1nventioh in orde'r to screen for new drug targets. The emphasis of pharmaceutical research activities has shifted toward the purposeful discovery of novel chemical classes and novel molecular targets. This change In emphasis, and timely tochridcgical breaktkrougi~s (e.g molecular biology, laboratory automation, combihatorial chdmistry) gave bi~h to high throughput screening, or HTS, which is now widespread throughout the biopharraaceutical industry.
High throughput screening involves several steps: creating an a ssay that i$ predictive of a particular physiological response; automating the assay so that it can be reprqducibly performed a large number of times; and, se,quEantially 4 sting samples frorii a chernical library to identify chemical strUctures able to 'hit' tfte-asoa, suggesting that such .struetures might be capable csf p'rovoking the int6ndq4 physiological response. Hits from the high throughput screen are foliriwed up in a variety of secondary assays to elaminate artifactual results, phrtieulsrly toxic compounds. Thus, th-e assays used in high throughput sare.etYs are iptended to c'letect the presence of chemical samples (e.g. compounds, substances, rriol.ecules) possessing specific biological or biochemical properties. These propertle~ are chcisen to identify compounds with the potential to elicit a spec!fic hiological response when appliad In vivo. High throughput screens identify both agents thst ban be used as drugs themselves and in addition, drug candidates that will Ultimately be used as drugs. A compound of a certain chemical class that is fdund to have some level of desired biqlogical property in a high-throughput assay cari then be thP basis for synthesis of derivative compounds. Cell based assays utilise intact cells in culture.
Examples of such assay include luciferase reporter gene assays and calciupi flux assays.
A particularly powerful method of pertorming a cell-based assay suitable for the identification of differentiation modulators and regprierative drugs, 'Is describeo in the -present inverjtibn, is to determine the differentiation status of progenitor colls by monitoririg a genetic marker of differentiation. The marker may be endogenous, such as ah expressed nucleotide sequence or protein that is present specifically in the differentiated Gell type bu] not in its progenitor, nor In any oth6r cell present in the cell populatioh used for screening. The marker may also be-ah exogenous marker (i.e. a rp-porter geng) which is introduced into the cell population used for scre!ehing in such a yiay that it is expressed specifically in the differentiateo c,ell type ~ut not in its progonitor, nor in any other_ cell present irt the cell population used for sareening.
Examples of exogenous markors are enzymes. such aO ;Lpe Z, or non-enzymic markers, sUch as the fluoj-escent proteins (e.g. GFP; YFP ptci), gr cell surfacP
ahtigen's which are not normally expressed in a particular c'eli lineage or which ara erpitope-m'oilified or from a different species. A t'ransgene or exbgenous m~'rker gene ?with associatod transcriptional control elements can be expressE6 in a manner that reflects a pattern representative of an endogenous gerie(s). This can be ~chieved by pssociating the gene with elements of a cell type-specific promoter, or by integrating I
the transgene ihto a particular locus which is expressed in a cell-type specific manner (e.g. see European patent No. EP 0695351).
In pne embodiment, the urtdifferentiated cell type used as the starting rnaitArial in the present irivention is modified to express two rxiarkers/reporters (e.g. botYi.
GFP and YFP) such that one marker indicates the presence .of the progenit or- cell type submitted to the HTS process, and the other marker indicates the presence of the di .fferentiated cell type produced- by the addition of a suitable modulator.
A number of suitable reporter gene systerns_and cellular screening assgys, including dual reporter systems, are disclosed in. reviews (and refereni;os ther~in) by Hill et af.
[Cu7rrent Opinion in Pharmacology (2001) 1:526-532] and by Blak'e [Current Upinion in Pharmacology (2001) 1:533-539] all of which are ihcorporated herein in their entirety.
Prbgenitor cell A grogenitor cell is any somatic cell which has the capacity to gerierate fully differentialted, functional progeny by differentiation and proliferation.
Progenitor cells include progenitors from any tissue or organ system, includirjg, but not liini-ied to, blood, nerve, muscle, skin, gut, bone, kidney, liver, pancreas, thymus, arld the like.
Progenifor cells -nay be distinguished from differentiatdd cells (ie. those cells wf~ich Inay or may not have the capacity to proliferate, i.e., self-replicate, hut which are unable to undergo further differentiation to a different cell type gnder r16rmal physiological conditions). Progenitor cells may be further distinguished from aUnormal bells - such as cancer cells, especially leukernia cells, which preilif6rate (self-replicaie) but which generally do not further differentiate, dospite afipeong to be immaturo or undifferentiated.
Progenitor cells include all the cells in a lineage of differeni-iation and p(oliferatibn priqr to the most differentiated or the fully mature cell.
A'sccording to orie embodiment of the present inventionf a distincti6n Is drawn between a 'stem cell' and 0 'progenitor' cell. Stem cells are typically pluripdtent and ~nultipote(it and can give rise to a number of lineages. Progenitor cells, <<nd in paiticular lineage-committed progenitor cells, are only c-opable pf i?roducing the cells of a singie lineage. Hpnce the developmental potential of progenitors is typically rnore restricted than that of stem cells, A second important difference betWoen stem pells and prog6nitor cells is that stem cells are capable of signifit:ant amplification -under tfle correct culfuring conditions they carl djvide indef I initely -wherea s tirogenitor cells have a limited proliferation capacity. These differences mean that, for instanoe; it is possible to reconstitute We haematopoiefic sy;;tem of'an anirl'ia1 using_ haematcipoietic stem cells but not haematopoietic progenitor c'ells.
By way of example,. production of mature, functional rod blood cel.ls result$
trom proliferatibn and differentiation of 'unipotential progenitot-s,' i.e., those -progenitors which have the capacity to make only ono type of one. type of blood cell.
Various other hematopoietic progenitors (HPCs) have been characlerized.
HPCs consist of many subclasses including pluripotent gtem cells, lymph'id stem cells, CFU-GEMM colpny forming unit granulocyte, erythroid, macroph(age nr megakaryocyte, BFU-E, CFU-E, CFtJ-Meg, CFU-GM colony fbrming unii granulocyte or macrophage, CFU-EoS colony forming uniteosinophil, progenitor B cells and progenitor T cells.
Stem Cells Stem cells are described in detail in Stem Cells: Scifgntifie Progress and Future Research Direbtions. Department of Health and Hurnan Seryices. June 2001.
http://wWw.nih.gov/news/stenlcell/scireport.htm. The contents of the report are herein inco'rporated by referende.
Stem celis- are cells that are capable of differentiating. to form at least onp and sometimes many specialised cell types. The repertoire of the different cells that can be formed from stem cells is thought #o be exhaustive; that is to say it includes all the different cell types that make up the organism. Stem cells are present throughout the lifetime qf an organism, from the early embryo where they are relatively abund~nt, to the adult where they are relatively rare. Stem cells present in many tissues of adult an~rnal's are, important in normal tissue repair and honieostasis.
The existence of these cells has raised the possibility that they cbuld provide a means of geperating specialised functional cells in vitro that can be transplanted into humans and replace dead or non-functioning cells in djs,ea~ed tissues. The list of diseases for which this may provide therapies ihcludes Parkinson's disease, diabetes, spinal cord injury, stroke, chronic heart digea~e, end-ctage kidney diseasse, 'ivdr failure and cancer. In order for cell replacembnt theraoy to becorne feesible a numher .of major breakthrau.ghs in stem cell researcb are i-equired, iniauding imlprovemeylts in the growth of stem cells, differentiaf ion of st6m cells and a~~~oidance of imm~rnological rej-ection of stem cells.
For this teasop, alternative approaches.to exploiting stem cells for therap.y are being considered. As described herein, methods are disclosed for' the discovery of cam-pounds which may' be developecl- into drugs (eg. i'egenerative drugs) that cause endogepous s~em cells to regenerate tissues of the body, Types of stem cell There i$ still cbnsiderable debate about what constitutes e stem cell, lioweve~ for the purposes of this discus'sion a key characteristic is the ability to differentiate into a differerit cell type. An optional characteristic is the ability to self-rene~N, in eerkain cases indefinitely, alloWing amplification of the cell numbers. Excjlimples 4f ste~n cells are given beloW.
Different stem cells have differing potential tp form various cell 6es:
sperrpatogonial stem cells are unipoterit as they naturally produce only spermatozoa, whereas haematopoietic stem cells are multipotent, and embrycinic steiri cells are thought to be-able to give rise to all cell types and are said to be totipotent or pluripotent.
To date three types of mammalian pluripotent stem cell have been isolated.
These cells-can.give rise to cell types that are normally derived from all thr-ee gerrh layers of the embryo (endoderm, mesoderm and ectoderm). The three typ+es of stem ceil are;
qmbryonal carcinoma (EC) cells, derived from testicular tumours; erribr*ii;
stem (ES) c-ells, -derived from the pre=implantation embryo (normally the blastocysi); and embryohic genn {EG) c>rlls derived from the post-implantption embryo (notmally cells of the foetus destined to become part of the gonads). These cells are receiving particular attention in the effort to ditect differentiation, precisely because they are pluripotent Stem cells are also present in the adult organism. An adult stcrn cell is an undifferentiated cell that pccurs in a differentiated (specialised) tissue, renews itself, and can differentiate to yield lnore specialised c0s. Recently it hi-~ is been sho,Wn that adLilt stem cells are capable of plasticity, that is to say they can diffdrentiate tb yield c.e.ll typos that are not characteristic of the tissue iri which they reside, hor indeed of the germ layer from which that tissue originates For example, it has been showri that blood stem cells (derived from mesoderm) can differentiate into neurnne (ilormally derived from ectoderm). Toma et al. (2001-, Nature C~ll Biol. 3, p778=784) f'lave recently desirihed the identification and isolation of a n~w type of ste~n -cell that was _84ived from- the dermis of the skin. These stem_ celis Were termed skin;
derived precursor (SKP) cells-. Thd S-KP cells could be induced to differentiate by cuiturino on _poly-lysirib and varyiny the concentration. of serum In thF, cdlture rr-11E:dium. In the absenpe of serum they differentiate into neurons arid g(iai cells; vvith qiiditi ~un of 3%
serum they differentiate into smooth-musele cells; and incret'asing the serum to 10a/o causes tlie SKP cells to differentiate into adipocytes. Adult stem cells heve so far been reported in tissues as diverse as the nervous system, the bone rrarrodv and blood, the liver, skeletal muscle, the skin and digestive system.
In addition to the adult stem cells there are numerous types of progenitor or precursor cells, as described herein. These are cells that alrp partially restricted in their differentiative potential and occur in probably all of the tissue's of the body ~ they are capable of differentiating but differ from steM cells in that their repertciire is riot as broad, and by definition they are not capable of self-reraewal.
Recent evidence even sua.gests that differentiated cell types a~e qapable of chqnging phenotype. This phenoinenon, -termed trarrsdiffereritiation, is- the -conversion pf orMe differentiated ceil type to another, with or without an intervening cell divisior). It was previously generally accepted that the ter'rninal differentiated ptate is fixed, but it ip now clear that-differentiatiomcan sometimes 'be reversect or altered. In vitrb protocols are now available in -which cell lines can be induced to transdifFerentiate (see Shen, Slack & Tosh, 2000, Nature Cell Biol.vol 2, p. 879-887; Horb et al, 2003, Current Biol. Vol 13, p105-116). 'Finally, there have been reports of specialised cbfl types that can de-difFerentiate to yield.stem-like cells with the potential to differentjate. into further cell types.
Stem cell gro*h and differentiation An important property of stem cells is their ability to djvidle symmetrically in culture, giving rise to two daughter cells that are exact copies of the stem cell from Which they were derived. This allows stem cells to be exparided id culture in their undifferentiated state, producing enough material for scrpenirig 'purposos, biological studies or even cell therapy. The means by which stem cells are able to do this is understbnd#ly the subject of intensive research, yet_fe* of the factors that prdmote Mem cell renewal are known. Typically, pluripotent sterri cell lines are rnaiiitairied on mitotically inactive feeder layers of -fibroblasts, oir medium conditioned by suclY cells.
It 19 absumed that feeder cells- remove/neutralise some unkncjwn factcir frorim the cul{ure medium, and/orthey pr-ovide a factor that supp'resses the differ-ehtiatioh_and promotes the seJf-renewal of stem cells. One such factor-is leukaemia.
inhibitory factor (LIF), a member of the cytokine family related to IL =6y which is capabte- of p'romoting mouse ES cell self-renewal in the absence ;of f0eder cells. Stem cells grown ig the absence of feeder cells (and/or LIF) oftea differentiatq I :
spdntaneously and haphazar~ly, producing a-mixture of differentiated pell type~. More reeently, ES
c,ell lines have been. produced that are able to grow in feedeP free culture arid iatlder defined conditiohs.
The factor~ that influen~e stem cell self-renewal may eitheF be stimulatoiy or inhibitory and may function extracellularly or intracellylai'ly, In the ca~e of the secreted factor LIF, it is known that its extracellular receptor is gp 130, and that activation of thi's protein is sufficient for inhibiting murine ES c--ll differentiation. Within the cell, a cruci6l downstream effector of gp130 is the signsl trahsducer arld activator of transcription-3 (STAT-3). Another molecule which is porticularly inipoilant iri rrmaintaining stem cell pluripotency is the transcription factor Oct-a=, which when Oownregulated artificially leads to the loss of the plu-ripoterit st6te In ES
cells Gr rn~cd.
Other signalling molecules that naturally inhioit ES cell self-ren'ewal, such a6 the mitogen-activated protein kinases, have also been elucidated. A rnajor= goal of stem cell research will be the discovery of- natural and- syrithetuc .fiictQr~, d'rugs, poly,pefitides, genes, oligonuclootides, tissue culture media and conditions, specific conditioned media-, feeder cells, and other stimuli that have the effect of promotigg the expansion ahd retaining the differentiation potential ef vai=ious- types of stem celL
This inciUdes adult stem cells, which at present have not been expanrted appreuiiably in cell culture.
The second great challenge of stem cell research is tq direct the differentiation of stem cells to particular cell types which are functional, can replace ceII.S
lost in various disease states, and result in a positive clinical outcome. Coaxing stem cells to begin differentiating is actually a fairly straightforward process. For instance, ES
cells removed from feeder cultures and grown to confluonpd on an adherent substrate will begin to riifferentiate spontaneously. Similarly, ES cell's r~';moved from feeder cultures and grown on a non-adherent substrate will form embryciitj b6dies -clusters of pndifferentiated and partially differentiatpd cells from all three -germ layers.
These cells can be subsequently dissociated and plated_ in monolayer cuature, and exposed tp factors that -promote dire-cted- differentiation. Cuitures~
exposed to. these factors--are more likely to be populated by-fewer types of d'ifferentjated cell, dompared to embryoid bodies or untreated cultures of diffbrentiating. cells which comprise mixtures of many different cell types. Nevertheless,-few if any conditions have-been devised thus fal- that produce substantially pure cultures.-of c;iifferen'tipied cells. in addition it is not clear if alny of the prbtocols devised for stem cell differeritiation yield dell.s tl~at are suitable for cell replacement therapy - it may be that the cells haue not terminally differentiated into the precise phenotype required, or that tke differentiated cells are rio longer viable in vivo.
The factors that have been iased to induce directed differehtiation bf stem cells inciude; retiiioic acid, epidermal growth factor (EGF), ijone morphogenic proteins tBMPs), basic f'broblast growth factor (bFGF), activin-j,4, transfqrming growth factor be.a-1 (7FG R-1), hepatocyte growth factbr, nerve growtk f6ctor, sonic hE;dgehop (SHH), interieukin-3 (IL-3), interleukin-6 (IL-6), granulocytO macrop~age colony stimulating factor (GM-CSF), erythropoietin, vitamin D3; dexamethasone, (3-mercaptoethanol, butylated hydroxyanisole, 5-azacytidine, DMSO, insulin, 'thyroid hormone (T3), LIF, foetal calf serum, vascular endothelial grovqth factor (VEC-lIF), steel factor, variations in oxygen concentration, ascorbic acid, fi,glycei-ophosphate, nicotinanlide, platelet derived growth factor (PDGF), cAMP, various cell adhesion moleculE;s and substrates, and others. In addition to these defiped factor';, it is lilcely that undefined extracts, such as conditioned media, humpn, and arhiriial iissue homogenates, or plant eztracts can be used to dlFeat stem cell differentiation.
Prpgressjve fractionation of tliese und-efinod extracts may yteld a.ctive fracti4ns~ or Oven pure bomponents with high -potency. These factors can ne added t6 the g~owth medium used in a particular experiment, either alone, or in combination, t~r iFy a defined &der which is crucial to the experimental result.
Many systems that have been devised for the differentiation of 'tem cells in v6q are cornplex multi-stage procedures, in which the precise riature of the varibus steb's, as well as the chronology of the various steps, are irhportant. For instanSe, Lee et el (2000, Natul-e Biotechnology, vol. 18, p 675-679) used a five sfage prot( I
Jcol to d4riv~
dopaminergic rieurons from mouse ES cells: 1) undifferentiated ES cells were expahded on gelatin-coated tissue culture surface in Eg bell medium in the presence of LIF ; 2) eMbryoid bodies were generated in suspension cultures foi* 4 days in ES
dell- med'rurn; 3) nestin-positive cells were selected firom embryoid bodies ii] ITSFn medium for 8 days after plating on tissue-culture surface; 4) neOp-positive bells were -expanded for 6 days in N2 medium containing bFGFAarninin; 5) fnall~ the PxOnded neuronal precursor cellS were induced to differentiate by withdrpynring bFGF
from N~-medium coritairiing laminin.
In a second -eicample:-of serial cell culture, Bonrrer-Weir et al. [Proc.
fyatl. Acad. Sci.
(200Q) 97: 7999-8004]- derived irisulin producing cells from human pancreatic ductal cells by: 1) selecting ductal cells over islet cells by selective ;adhesiQn on a solid surface in the presence of serum for 2-4 days; 2) subs6quently withdi-awi0g serum and adding . keratinocyte growth factor to select for ductal epithelial cells over fibroblasts fqr 5-10 days; and 3) overlaying the cells with the extracellular matrix preparation 'Nlatrigel' for 3-6 weeks.
In a further example of serial cell culture, Lumelsky et al. fScierieb (200~1) 29i2: 1389-1394=] derived insulin sec'reting cells by directed differentiatioh af mouse embryonic stem (ES) cells by: 1) expanding ES cells in the p'resence of LIF for 2-3 day~; 2) generating ernhryoid bodies in the absence of I-IF (Jver 4 days; 3) selecting nestin-positive 6ells using ITS1=n medium for 6-7 days; 4) expanding pancreati'c endocrine precursurs in N2 medium containing B27 media supplement and bFGF for 6 days;
and 5) ihducing differentiation to insulin secreting cells by withdrawing bFGF
and adding hicotinamide.
However it Is not -only the sequence and duration of the various 'steps d thfe series of additiori of different factors that is important in the determination 6f cell diffe'rPrrtiation.
As embDronic development is regulated by the action of _gradients jof signalling factors that impart positional information, it is tQ be expected that the concpntration of a single signalling factor, and also the relative conceqtration of two or m'ore fac'tors, vtiriIl be irnportant in specifying the fate of a cell population in vitro and' in viv6. Factor concentraltions vary during developrrient and stem cells responc] differe,ntly tb different concentrations of the saMe molecule, For instance, 5terri ceOls isdlate'd from the CNS of late stage embryos respond differently to different cancentration$
of ~GF:
low concentrations of EGF result in a signal to proliferate, while 1ligher conoentrations of EGf' result in proliferation and differentiation to astrdcytes.
Many 6f the factors that have been found to influenpe self-rejiewal and differantPation of sterri cells in vitro are naturally-occurrprig molecul.es, This is to- be expected, as differentiation is induced and contralled by signalling molecuies--and _reee6tor-s tkiat pct along signal transduction pathways. However, by the san'letd-ken, it is likely that many synthetic. compounds will have an -effect on stPr--n celi differelntiaitjon. Such synthetic compounds that have high probability e.f. iritPractir-;g wifh cellular targets within signallirlg and signal transduction pathways (so called' drugable targets) are routinely synthesised, for instance for drug screening by pharmaceutiaal Cpmpanies.
Once knowrr, these compounds can be used to direct the differontiation of stern cells ex vivo, or can be administered in vivo in which case th-6y wotlld e'ict on resident stem cells in the target orgah of a patient.
Common variables in tissue culture Ir4 Oevelopino conditions for the successful culture of a particular cell typo, o-' in order to achieve or modulate,a cellular process,. it is often important to consider a vaflety of factors.
One important factor is the decision of whether to propagate the cells in suspensiori or-as a inbriola~er attached to a substrate. Most cefls pref~r to adhere to a4ubstrate OIthough some, including transformed cells, haematopoietic cells, and cells from ascites, dan be propagated in -suspension.
Assuming the culture is of adherent cells, an important t'actor is ttie choice of adhesion substrate. Most laboratories use disposable p(astics as subs,trates for tissue culture. The plastics that have been used include polystyrehe (ihe most common type), polyethylehe, polycarbonate, Perspex, PVC, Teflon, 6e4lo~hane and cellulosp acetate. It is likely that any plastic can be used, but many of these Will need to be treated to make them wettable and suitable for cell ~ttachment.
F~rthermore it is very likely that any suitably prepared solid sub'strate can be used to provide a support for cells, and the substrates that have been used to date include glass (e.g.
aium-borosilicate and soda-lime glasses), rubber, syiitretic fibre.~, pol~imerised dextrans, metal (e.g. stainless steel and titanium) and others.
Some cell types, such as bronchial epithelium, vascular endothelium, s,keletal rnii~cle anri neurons require the growth substrate to be -coated with bioiodical proqucts, usually extrecellular matrix-materials such as fibronectin, cdllaben, laminin, polylysine pr others. Thie growth substrate and the method of application (Wet or diy-poating, or gelling) can. heve ar.'i effect on cellular processes such as the growth and differeritiation character-istics of cells, and these must be de#errni,neci empiricaat.y as disbussed ebove.
Probably the most obviously important of the variables In cell culture is the choice of culture medium and supplements such as serum. These provide an aqueous compartment ('or cell growth, complete with nutrients arid various factors, some of which have been listed above, others of which are poorly defikied. Some of these factors, are essential for adhesion, others for coriveying informEltion (e.g.
flormones, mitogens, cytokines) and others as detoXificants. Co'rrirrionly osed liedia include RPMI 1640; MEM/Hapk's salts, MEM/Earle's salts, F12, DMI~IA/F12, ~.15, MCDB
153, and others. The various media can differ vyidely in their constituents -sbi'ne of the oommori differences include sodium bicarbonate honcentration, concEntration of divalent ions such as Ca and Mg, buffer comp.osition, antibiotii,s, trace elenients, nuc;leosides, polypeptides, synthetic compounds, draags, etc. It is weJl known that different media- are selective, meaning they promote the gr6wth of bnly sqme cell types. Mqdia supplements sU4h as serum, pituitary brain arjd othef extracts, arP qften essential for the growth of cells in culture, and in addition are freque~itly responsible fi?r determining the pheiiotype of cells- in culture, i.e. they are calpable of deter-rnining cell su.rvival- or directing Oifferentjation. The role of supplements in cell processes suqh as di -fferentiation is complex -and depends on thpir coricentration, tpe iime point at Which they are added to the cultur?e, the cell type and thddiurri tased.
The updefined nature c-f these supplenietyts, and their potential to affect tl~e cell pr'iendtypb, havp motivated the development of serum-free media. As with a1i mledia, their development has come about largely by trial and error; as h6s been discOssed above.
The gas phase of the tissue culture is also important and its bbmpositioin and volume vbhich should 4e used can depend on the type of inediurri used, the amouht of buffering requifed, whether the culture vessel is open or seclled, and vihether a particular cellular process needs to be modulated. Common vdriab(F:, include concentiation of carbon dioxide and oxygen.
bther conditiops important to tissue culture include the chdice of culture ve~sel, amount of headspace, inoculation -density, temperature, frequ~/nc~:y of ineaib charig-es-, treatment witl=r enzymes, rate and mode of agitation or' stiring.
Varying the cetl- culture conditions is therefore a method of achi~-vi~'ig a-dosired celiular process. One aspect of the invention- recognises_ that variation of thP cell culture conditions in a serial manner can be a highly effdctive niethod foh achieying_a cellula,r effect. In various applications, for instance in $tUdles of cell differentiation, it will often bd the case that a specific series of differpnt tissue cUlture co'nditioi ns ol-e required to effbct a cellular process. The different conditions may include additions or withdrawals to/from the media or the change of media at 4pecM4 tim6. 9bint,-:,. Siach a set of conditions, examples of which are given below, are commonly qeveloped by tridl end error as has been discussed above.
Formation of cell units An irimportant aspect of the present invention is that groups of cells (cell colonies) can be growp in cell culture under various conditions and that the colony c~n largeJy rpaintain its intedrity undervarious copditions, when disturbed, 6nd when mixed with oth~r colonies. Such groups or colonies are referred to horein as cell ianits.
Formation qf cell units may be achieved, by way of illustratioh, py growing cells as' adherent ctyitures-on-selid substrates such as carriers. I-f ~elt proliferation oocurs-after seeding on the- carriers, the daughter cells will attach -on the- same corriE:r and form part of the sanie colony. In -general, live- adherentcells do not readily -dissqciate from their -grovirth substrate,.ans1 so the integrity of the cell cqiony persists despite any mechanical rnanipulation of the carrier, agitation of the culture. medium, or t'ransfer into anotheh tissye culture system. Similarly, if at any time multiple carrieFs are placed iq the ;same u6ssel (e:g. beads are pooled) then there will be rio substantial transfer of celfs fr6rn pne bead to another.
Ohe advantage of growind Colls in units or eolonies is that if a unif is placed 'sefially in a set qf different tissue culture media, then all the cells comprisind the coli)ny will have been exposed to -the same series of culture conditions, in the same order and for the same period of time.
Another of the advantaRes of this m.ethod- is that tissue culture can be miniaturised:
relatively few cells are requited to colonise a micrcicarrier bead (see below) coirnpared to even the smallest tissue culture flask.
Growin-c~'- cells- in units that are not necessarily themselves aCiherent to the tissue culture vessel has the further advantage that-individual eolonjes can be rer-hoved at wili- and ttansferred to a different culture vessel'. This is partjcUlarly impc)rtaht in th,'e present invention as it allows for the transfer of differentieteo cells comj~rising progenitors to rriicrotitre plates suitable for drug screebirlg. Thus the Orooor)iiors can be prepbred in bulk - such as by a method previously detern-lined Using the techniq6e disclosed in WO2004/0~1369, and then the same cell units transf6'rred essentially by iiquid transfer into the HTS platform, This (i) greatiy facilit'ates tl~e HTS
procedyee, (ii) maintains the 3D (multi-) cellular organisation of Onits which may be integral to further differentiation, (iii) and obviates any disfioci6tion of cellular ordanisation which may in itself cause cellular differentiation.
~2 A furthar advantage of growing cell units formed on carrlers is thet cell cultu~e can be scaled up. Growth of stem cells ori carriers offers a way of scalinp up Krroduction to provide enough material for high throughput screening. Scale up qf such cell cultures may require at least I g (dry weight) of microcarrier - such as 1 pg, 50g, or more.
Another important advantage of forming cell units on solid substrates is that the ,gubstrate - and therefore the attached cells by reasori of association - can be labelled by various-means:
Glass 'spheres of 3mm and 5mm have been widely used as celf adhesi6rl substrates, particularl~ in glass bead bioreaefors-(e:g. such as manufactured by Meredos Gmbh) used for the scale-up of cell cultures. These beads are typically uwed in nackpd beds rather than batch culturO, to avoid mechanical damage to the adhereht cells.
Though such subptrates are suitable for the- purposes of this invention, other barriers described below rnay be even more suitable.
In particulari when cells are grown on smaller carriers they can be treatec~
as a , ;~uspe , rision Jculture. Import2ltitly, a common method dt growing cells on smafl carriers is referred to as microcarrier cell culture (see 'Microcarrier dell pulture, I?rinciples and Methods', Editidn AA, available from Amersham Biosciences (18-1140-62~);
he'rein incbrporaied In its entirety by reference). Microcarrier culturhs are .useq commercially fo-r antibody and interferon production in fermenters of -bp to 4ob, d. litre . A Wariety of -micl ocarriers is available, ranging in shape and size aiid made of difFerent materials.
iVlicrocarrier bbads made of polystyrene (Biosilon, Nune), glass (Biogiaas, Solohill 'Eng), collagen (Biospheres, Solohill Eng), DEAE sephadex (Cytcidex-1, Pharnlacia), dextran {Dormacell, Pfeifer & Langen), cellulose- (DE-53, Whatman), gelatih (Gdlibead, Hazelton Lab), and DEAE dextran (Microetex, Deactran Prod.) amoiigst others ere commercially available. Thes.e-carriers are well characterised-in-terms of the specific gra'vity of the beads, the diameter,and the surface area available fgr cell growth. In addition a number of porous (micro) carriers are pvailable witfl greatly increased surface area for cell growth. A further charactei'iotic of these porous carrier~; is that they are suitable for growth of both anchorage dependent 0611s, as well as for suspension cells which are carried by entraprn6nt in the network of ppen, interconnecting pores. Porous carriers are available in materials sucfl as pellatin (Cultispher G, Percell), cellulose (Cytocell, Pharmacia), polydthylyne (Cytolirie 1 arid 2, Pharrna~ia), silicone rubber (Immobasil, Ashby Sciehtifjc); collagen (Mici osphere, Cellex Bioscienoes), E(rld glass (Siran, Schott Glassware). T'hese carriers are ~ariously suited to stirred, fluidised or fixed bed culture systoms.
A~s the Ohysicel properties of carriers are well known jt is -easy to cElIculate the number of carriers used in an experiment. Some of the carriers described-and many besides are available as dried products which can be accurately weighed, and subsequently prepared by swellirig in liquid medium. In additiori the number of cells used to inoculate a microcarrier culture Gan be-worked-out-antl varied:
For.ins,tance, e culture of Cytodex 3 (2 g/Iitre) inoculated at 6 ceilS per bead will give a culture-containing 8 million micrpcarriers, on -which 48 million cells/litre ari~
grovyn aL a density of 5x1 Q4 cells/cm2.
If required, harvesting of cells grown on microcarriers, or lik)er~tion pf labels from microcarriers (see below), can be achieved by erizymatic detalchment of cells, and/or by digestion of the carrier where applicable: gelatin corriers can be soliabilised by trypsin and/or EDTA, collagen carriers using collagenase and dextran cbrriers using dextranase.
In addition to solid or porous hnicrocarriers, cells rhay bd grouped by immurement, i.e.
confined within a medium permeable barrier. Membrane culture systems have been developed where a permeable dialysis membrane retains agroup of celJs,. but allows the culture medium and. its constituents to exchange freely ~vith- the inner and outer compartments. Cell culture in hollow fibre cartridges has-also been 4_velbped, and a rnultitude of fibres-and even turn -,key systems are ciorpmercially availaple (e.g: from .An'ii,r,on, Cellex Biosciences). Cell ancapsylation in semi-solic) matrices has Eflso been developd, Virhere cells are immobilised by adsorptiori, coyalent bondilaq, c~os.I~Iinkin-g or entr-apment-in a p-olymeric matrix. Materials that have 6elen used include gelatin, pcl.kl.ysine, alginate and bgrarose. A typicai protocol, is to Tix ~% dgar-o'se- at 40 C-with a suspension of cells in their normal growth medium, to emLiisify th-e mixture using an eqUal volume of paraffin oil and to cool in an ice bath, producing spheres of 80-2004m diameter. These spheres can be separated frorp the oil and transferred to mediurri in ia tissue culture vessel.
Cell entrapment is a simple method for the immobilisation of groups of cells, akin to the use of microcarriers or porous substrates. A simple technique is fo enr'nEsh cells in cellulose fibres such as DEAE, TLC, QAE, TEAE (all available frorn Siprna).
Other more sophisticated devices are ceramic cartridges which ere suitable for suspension cells, as in the Opticel culture system (Cellex Biosciences).
One skilled iri the art will envisage, in addition to the above rnethods of creating cell units, other methods of creating groupings of cells irtriudirig farming 3G
culiurOs of cells such as neural spheres or embryoid bodies, or using tissues and ihtlegd whole organisms such as Drosophila or C. elegans.
Cell- units, or the substrates of which they are comprised, can be associated with a particular facto.r including, but without limitation, _proteins, nucleic aciits or other chemicals such as drugs. Pre-conditioning of substrates can be achieved irr.
r~any ways, for instence simply by incubating the substrate with the fa6lor of interest, or by attaching the factor cbvalently or non-covalently to the subgtrafE. ~olubl(r factors can be incorporateo into dry materials by impregnation. This technique relies on th~ rapid ingress of I~quid, carrying soluble factors, into dry porous material that concomitantly becomes swollen and ready for use. Solid factors carr be incorpprated for example by mixing the factor in fibrinogen with thrombin solution, at whicti poini a fibrin clot containing the factor is formed. Multiple other ways can be envi'saged of assoeiating factor(s) With a cell group, in addition to impregnating, entrappirig or encapsulating the factor together with cellg.
A method for associating a cell group with a number of different factors is to pre-form cockteils of factors which are $ubseq.uently assobiated with a parficular +cell group. A
secend method would be to serially condition -cell groups in a nui-nber of factors.
Using dry-formulations of eell group growth substr'ates ab an example, this method would involve firstly partially swelling the substrate in a solution.
containing a first factor and subseq:uently further swelling the-same in a solutiop containirty a secohd factor, resulting in a substrate to-which both factors h'We beoomE associai-ed. By Oevising a s'=ystematic prottjcoi of assrociating cell grc;ups with differerit comkiinations of differOlt factors it will be possible to salmple the effect on the cell group of any combination of a set of factors.
Regardlesh of the method used to condition cell uriits with factqrs, the factors are taRen up by cells that coirprise that cell unit. Factors leaking into the growth mediuin are diluted to such an extent that their concentration falls below physiblog;cally relevant limits and they have no effect on any additional cell groUp to which they are expose~. The diffusion of the factor out of e.g, the substrate forming port of th~, coll unit is governbd by parameters such as the nature and dimexisiohs eif tb'e material, the mean pore diameter, and the molecular weight and poncentration of Ithp fdctor.
To. dalibrate the process if necessary, factor release can be measured by physical ~ssays such as HPLC analysis. or release of labelled factor into the mbdilam, oi~ by biological assays such as the dorsal root ganglion- outgrowth bioass8y for neurotroPhic factors.
Cvrr.'ibinatorial serial culture of cells The inventian further addresses the problem that cell cu!tqre tec:hniques involving a plurality of steps and agents are difficult if not irnpeissible to determine by conventional experimeritation, which in the -prior art has involved trial and error:
1=mpirical determination of tissue culture conditioris in complex, inuiti-stage procedures is not feasible in practice. Advantageousl~, the cultdr~ conditions required to differentiate a cell type may first be ideiitified using the methods described herein below.
(a) Split-pool cell culture Split-pool culturing allows cells to be subjected to a series of culfiure conditions, and exposed to a series of agents In culture media, in a systematid and highly productive manner and is described in detail in W02004/031369.
The first-step.in split-pool cell -cu(ture is to form cell units (particularly microscopic cell units) as-each cell unit constitutes an -easily handled unit that can be exposed to a variety- bf cell culture condi#i.ons. For- simpiicity, in this di$cussiori we-wiii assume that cell groupings are produced by growwing- cells in microcarrier culture, a9d~
thL--terms cel6 unit, cell group, colony and bead are used intetchangeabiy._ Howev4r, the methods desbribed are equally applicable to any clell gnit. A. particuld.irly efficient method for sampling a large number of cell culture conditiods is rFferred to as CUnibinatQrial Cell Culture or split-pool cell culture and in one ombodiment involves the serial sqbdividing and combining of groups pf cell units in order tq sanlple multiple combinations of cell culture conditions. In one asp c~ of the irivention the method operates by taking an ihitial starter culture (or dlfferent starter cultures) of cell pgits divided into X, number of aliquots eaich containing multiple beads (groups/colonies/carriers) which are groWn separately under different cditure coriditions. Following pell culture for a given time, the eell units can be pooled by combining and mixing the beads from the different aliqubts. This pool cah be split again into X2 number of aliquots, each of which is cultured unoer different ccmditlons for a period of time, and subsequently also pooled. This itOrative procedure_ of 'splittingj cuituring and.pooling (or pooling, splitting and culturing;
lepending bn Where -one enters the cycle) cell units allows systematic sar~ioling of meiny ciifferent combinatioris of cell culture conditions. The complexity of the experiment, or in other words the nurnber of different combinations of cell cuiture conditions testeo, is equal to the pr-pddct of the number of-different conditions (Xi x X~-x ..Xn) sampled_ at each round. Note that the step of pooling all the cell units prior ta p subsequent split car1 bc~
optional - a step in which a limited number of cell units are poolecl can have the same effect. The invention therefore embodies a number of r-elated meihod.,-of systematically sampling multiple combinations of cell cultyre coriditions where qrbup's of qell units are handled in bulk.
Regardless of the precise manner in which a diversity of cell culture conditions is sample6 by this means the procedure is efficient because multiplE cLall units can share a single vessel, where they are cultured under identical condition$, and it con be carried out using only a few culture vessels at any one time (the nutnber of cul#ure vessels in use is equal to the number of split sample's). Iri many respects the principle of this procedure resembles that of split synthesis of large chemical libraries (known as combinatorial chemistry), which samples all possible conibinations Of linkage between chemical building block groups (see for example:
Co.r~ibinetorial Chemistry, Oxford 'University Press (2000), Hicham Fenhiri (E8itor)).8plii-pool cell culture- can be repeated over-any number of rounds, and any nUmber of conditions can be sambletl at each round. So long as the nUmper of cell units (qr co+onised beads in this example) is greater than or equal to the nLmber olr different conditions sampled over all rounds, and assuming that the -splitting of cell units occur.s--totally randomly, it is expected that there-will be at least one cell unit that has bben cultured according to each of the various combinations of cultur6 conditigns sampled by the experiment. This procedi.ire can be used to sample growth or differentiaition condition for any cell type, or the efficiehcy of bjomolecule production (e.g.
production of erythropoeitin or interferon) by any cell type. Becpuse tli4 procedure is iterative, it is Ideally suited to testing multistep tissue culture protocols = for instaiice tha.se deccribed above in connectidn with stern cell differentietion. T'he ~ariable's which can be sampled using this technique include cell type, cell groupirig (e.g.
microcarrier culture, cell encapsulation, whole organism), growth suhst'raie (e.g.
fibronectin bn microcarrier), duration of cell culture round, temperature, different culture media (including different concentrations of con$tituents), growth factors, conditioned riiddia, co-culture with various cell types (e.g. feed6 ' r cells), animal or plant extracts, drugs, other synthetic chemicals, infectiori v~ith viruses (incl.
ttansgenic yirUses), addition of transgenes, addition pf ~ntisense qr anti-gerMe rnolecules (e.g. RNAi, triple helix), sensory inputs (in the case of organisms), electricai, light, qr red-ox stimuli and others.
In._one embodiment, the culture conditions required to differentiate tho fr:;t cell type pre first ielentified* in a method comprising the steps of: (a) providing a first set of groups of cell uriits each comprising one or more cells, and exposing said groups to , c#esired culture conditions; (b) subdividing one or more of said groups~ ~to create a further set of groups of cell units; (c) exposing said further gr)ups #o fur~er desired culture conditions; (d) optionally, repeating steps (b)-(c); and (e) assessing the effect on a-given cell Unit of tha culture conditions to which it has been exposed.
In another embodiment, the culture conditions required to partially ot fully differentlate the first cell type are first identified in a method corriprising the steps o(:
(a) proyiding a first set of groups of cell units each comprisipg one or rYiore cells, and exposing said groups to desired culture conditions; (b) pooling two or m~ore af said groups to forrn at least one second pool; (c) subdividing the second pool to crelate a further pet of groups- of cell units; (d) exposing said further grolu.ps to desired cu'ture conditioms; (e) optionally, repeating steps (b) - (d); and tf) as~essing tfjq pffecf on given cell unit of the culture conditions to which it-has been-exposed.
Suitably, cell units that are partially differentiated are isolated -and, used in the rnethotfs described herein.
5uitably, cell uhits are labelfed and the label(s) reflect(s) the culture , condrtiohs to which the cell unit has been exposed. The label may be spatially encodad. The label may be selected from the group consisting of a virus, an oligpnucleotide, a peptide,_ a fluorescent compound, a secondary emine, a halocarbon, a mikture of stable isotopes, a bar code, an optical tag, a bead, a quanturn dot and a radiofrequency encoding tag or combinations comprising at least two of these labels.
Two or rriore labels may be selected and used in combination to label a cell unit.
Suitably, the ceils are cultured in cell units, each cell unit comprising one or more cells. ~'he cell units may be single cells. Each cell unit may comprise one or more cells adherent -fo or bounded by a solid substrate. The solid 4ubstrate may be a microcerrier qt bead. The solid substrate may even be a well or mediurn-pei'meable barrier.
In one embodiment, the eul.ture condftions are media to which -~he cell is expqsed.
Suitably, the media contain one or more specific agents which influence a cellular process.
The cell culture- conditions may comprise culturihg at one -or n16re Specific tem-perattJres or partial pressures of oxygen or -carbon dioxide. Tho cell culture ponditioris may comprise culturing on o-ne or more speeific substrates.
(b) Split-split cell culture The purpose of performing split-pool processes on cell units is to systehiatically expose these to a pre-defined combination of condition's. The person slfilled in the art will conceive of many different means of achieving this outcor~ie. In addition ttb split-pool processes and variations thdreof, it is worthwhile briefly discussipa ,plit-split proi;esses. A split-split process involves subdividing a group of cell unitq at least twice, Withqut intervening pooling of cell units. If split-split 0roces'ses are u~ed oV,'er a large number of rounds., the number of separate samples that ar'e gerierated increases exponentially. In this case it is irnportant to en.)pIoy some lev;l -of automation, for example the use of a robotic platforrri and sophisticated ~ample trat:kiny systerns. The advantage- of split-spUt steps is ,that (since cell upits are hot combined)- it is possible to -segregate lineages of the vatious cell units based on-thpir cell culture history. d.onsequently--split-split steps can be used to dedUcq if a particular cell culture condition is responsible for ainy-given cellular prucess and-therefore used to dediic,~- the culture history of cell units (explained in detail under 'Determinatiqn of culture history of a cell unit').
Predeter'rtlined protocols The splitting and/or pooling of cell units may be accdmplished totally randoT
ly or may follow a predetermined protocol. Where cell uriits are split andyor pooled randomly, the segregation of a given cell unit into any oroup is nbt preoete~mined or prejudiced 10 eny way. In order to result in a high probability that at leost one 'cell unit has been Qkposed to eech of the possible combin;atioris of cell culture p onditions, it is a6antageoUs to employ a larger number of cell units thari tho total nurr ,iber of combinations of cell culture conditions that aro beirig tested. Urider cE ' ~rtain circumstances it is therefore advantageous to. split and/or pool cell units according to a predetermined protocQl, the overall effect being. that adventitious duplibabons or omission6 pf combinationa are prevehted. Predetermined hanrlling-of ceil unjts can I;~e optionally planned in advance and logged on ii spreadsheet ar camputer probramrpe, and splitting and/or pooling operations executed usind automated protot;,ols, for instance robqtios: Labeifirrg of cell units-(see-belovV). can ~e by, any of a putnber of means, for iristance lebelling by RFID, optical taqging or spatial encoding.
, Robotic d-eVices capable 6f deter-tining the-identity tif a san;:i~.)le, -ciiid therefore parti{ioning, the samples according to a predetermined protocil, have been des,cribed (see 'C.ornLiinafiorial Chemistry, A practical Approach', Oxford University Press (2000), Ed H. Fenniri). Alternatively, standard laboratory liquid ha'ridling and/or tissue culture rbbotics (for example such as manufactured by: Beckman Coulter Inc, ~_ullertoii, CA; The Automation Partnership, Royston, UK) is capable of spatially encoding the identity of multiple samples and of addingr re1 moving or translocating these abcording to pre-programmed protocols.
Analysis and/or separation of cell units Following each rouhd of cell culture, or after a defined number of round~, the cell units can be ~jtudied to observe any given cellular proces that may hava been affected by the tissue culture conditions. The examples belov4 are illustrative and nbt infiended to limit the scope oT the inventipn.
Following each round of cell dulture, or after a--defineq nu~ber-of i odnas, tf1O qell uTtits can be assayed to determine uwhether there are members displayipg iricrelsed cell -prgliferation. -This can be achieved by a variety_ af techniques, for instance by visual inspection of the cell units under a microscopa, or b~ 9uantitating a marker product characteristic of the cell. This may be an ondogenous mdrker such as a particuiar DNA sequence, or a cell protein which cah- be detected by a ligand or aniibody, Alternatively an exogenous marker, such as green fluore,~,-,cent protein (GFP), can be introduced into the cell units being assayed to provid~ 9 specific readout of (living) cells. Live cells can be visualised usin'p a variety of vital stains, or conversely dead cells can be labelled using a variety of inethods, for instance using propidium Iodide. Furthermore the labelled cell units cpn be separated from unlabelled ones by a variety of techniques, both manudl and automafied, includirig affiriity purifibation ('panning'), or by fluorescence activated cell sorting (FACS) or broadly similar techniques. Depending on the application it rriay be possible to use standarct laboratory equipment, or it may be advantageous to use s.becialised-ipstrumentation. For instance, certain analysis and sorting iiistrumeiiil (e.g. sei?-Union Biometrica Inc., Somerville MA, USA) have flpw cell diameiers of trp to one miilimeter, yuhjch allows flow soiting of beads with diametei's Np to 500 riierons.
Thdse instruments provide a'reading of bead size and optical depsity as well as two ftunrescent emiesi-on wavelengths- from tags such as GFP; YFP Dr qS-red.
Softing speeds of 1$0,000 beads, per hour and dispensing into multi-vrell plates or into a bulk receptor ar-e possible.
Following each round of cell cttiture, or after a definei,i'number of rounds, the cell units can be assayed to deteroine whether there aro rimembers displaying a partidular genjotype or phenotype. Genotype determination cah be' carried odt us~ng well known tec;hniques such as the polymerase chain reaction (PGR), fluoresGnc; in situ hybridisatiop (FISH), DNA seq.uencing, and others. Phenotype ddterrpiriation can be carried out by a variety of techniques, for instance by visual inspection of 'the cell units under a rpicroscope, or by detecting a marker product characteristic of the cell.
This may be an endo.genous marker such as a particular DNA or RNA sequence, or a aell protein which can be detected by a ligand, conversion qf an enzyr~ne substrate, or antil~ody that recognises a particular phenotypic marker (For instarice se.e.
Appendix E of Stem Cells: Scientific Progress and F'uture- Research Dirbctioris.
Department of Health and Human Services. June 2001; appendices incor-ocirat>rd herein by reference). A genetic marker may also be exogenous, -i.e. one that has been iritroduced into the ceil- population, for example by transfect'on or viral transduction: Cxamples of exogenous markers -are the fluorescent proteins (e..g.
G,FP) or bell surfac.e antigens- which are not normally expreosed in a pprticular c6Il lineage or which are .epitope-modified, or from a-different sj~eci'es. A
transgene or exogenous maiker gene with associated transcripti6mpl control e4emdhts can be expressed in a manner that reflects a pattern representative of i~n endogpnous gene(s). This can b-e achieved by associating the gene with a minimal crll-type-specific prorrioter, or by integrating the tranagene intp a particular Ioc:us (e.g. see Europea'n patent No. EP 0695351). The labelled cell units cah be separated from unlabelled ones by a variety of techniques, both manual arid gutol-nated, in~luding affinity purification ('panning'), or by fluorescence activated cell sorting (FACS).
Nishikawa et ai (1998, Development vol 125, p1747-1757) used cell syrfape markbrs recogrtispd by antibodies to follow the differentiation of totipgt'pnt rpurine PS cells.
Using FACS they were able to identify and purify cells of tho haomatopgietic lirteage at various stages in their differentiation.
An alternative or complementary technique for enriching cell units of a particular genotype or phenotype is to genetically select the desired groups. This can be alchieved for instance by introducing a selectable markeP into the cell units, ahd to assay for viability under selective conditions, fpr lnstahce see Soria et 61 (2000, Diabetes vol 49, p1-6) who used such a system to select insulin secreting cells from differentiated E$ cells. Li et al (1998, Curr Biol voi- 8, p 971-974) identified heural progenitors by integrating the bifunctional selection marker/r.eporter Rigeo .(which provides fbr R-galactosidase -actjvity and G418 resistance)- ihtq lhe Sox2 IoOus bv homologou,s recombination iri murine ES cells. Since one of the characteristics of neural progenitors is expression of Sox2, and therefcire the iritegrated Marker genes, these cells bobld be selected from non-neuronal lineages by addition of G418 pfter inducing differentiation using retinoic acid. Cell viability cobid be determined by inspbption ulnder a microscope, or by monitoring P~gal activity. Unlike phehotype-kiased selectiori approaches, which can be limited by th8 availability of ah appropria-te I;gand or antibody, genetic selection can be applied to any differentially expre~sed gene, -IVlicrocarri6rs , , A variety -of microcarriers are available, ranging in shape and size and made of different inater'als.
By way cif example, ttre microcarrier may-be a porous microcarrier seleeted from the group i;orasi5ting of Cytopore microcarrier (eg. -a Cytopoie 1 microcar_rier or a Cytopor6 2 microcarrier), a. Cultispher microcarrier, a -~-Ui-t6pher-G rriic-mcarrier, a Cultispher-GL microcarrier and a. Cultispher=S- microcarrier, an Infqrmatrjx microcarrier, a Microsphere microcarrier, a S.ir.an microcarriei; 6nd a Midi-opqrous MC microcarrier.
By way of furthiBr example, the microcarrier may be a solid microcarrier =
5uch as a Pytodek microcarrier (eg. a Cytodex 1, Cytodex 2 or Cytodex 3 mii;rocarFier) a Biosilon microcarrier, a Bioglass microcarrier, a FACT III mici=~oqrrier or a microcarrier.
Microcarrier culture has sigrlificant advantages, including the scale-up of cultures, and also allows units of cells to be exposed to selected culttlre conditioiis as required in order to obtain the desired growth and/or differentiation conditions.
The surfaces pf the microcarriers may be further inodified by physical or Ghemical treatmerits, such as adsorption or covalent cross-linking of molecular eritities with a desired charge or other desired characteristic.
Cultisph pr microcarriers CultiSpher is manufactured from pharmaceutical grade poPcine gelatin via a process which yields a highiy crpss-linked gelatirl matrix with high me,chpnical and -thermal i,tability. Wheri used in ceN cultures, cells can attach to lpoth the external and the internal sutFaoes of the matrix. The increased surtade area of the matrix together with the proteotion from stress afforded to the cells in the interior of the matrix results in enhanced eell production capabilities. An additional advantage of the 6rotluct is thOt the matrix can be dissolved with prciteolytic enzymes resulting in the harvesting of cells with alrnost 100% viability, In one embodiment, the microcarrier is a Cultispher-G microcarrie~r.
Cultlsphe~G has a particle diameter of 130-380 pin, a volume of 12-18 rdl/g dry, a density of 1.04 g/ml with an average pore diatneter of 20 pm.
In order to 0repare and use Cultispher-G microcarriers, reference f;an be made to inter alia l3iotech. Bioeng. {2000) 6.8, 1 p59-70; Brii; J. Cancer. Suppl.
XXVII, S-78-S8? (1966); and the manufacturer's Website at www.porcell.se.
Cytopore, 2 rriicrocarrier Cytop'ore rrpicrocarriers are available from GE Heaithcare (preViousl~
Amersham) (www.microcarriers.com). Cytopore. is made of 1-00% cellulose, which is non-toxic to the cPlls and biodegradabie. It is positively charged-, due fia-= the N,N,-diethylatninoethyl groups. It has a very precise partiale oize distribtltiok and a network structure, the ratio of surface area to particle material is more than 95 to 1.
The network st'ructure enables stained cells to be cldsely observed wfiile they grow inside the mierocarriers. The typical particle diameter is 200-280 pm and effective surface area is 1.1 m2/g dry. The relative density is 1.03 g/ml, the average diainetor of pore openings is 30 pm and the volume is 40 ml/g dry. Iri order to prE:pard and use Cytopore microcarriers reference is made to intep alia Applied Microbiology and Biotechrlology (1997) 47, 4 p352-7; Cytotechnology (1999) 30 p143-147; Chinese Journal of Biotechnology (1999) 15, 4 p239-44 and Acta Otq-Laryngologica (2002) 122, 5 p541-5.
Cytppore 2 has been optimised for anchorage-dependent cel,s reqoiring a ch~rge density around 1.8 meq/g.
In some embodiments, the microcarrier is a porcine gelatin midrocarrie'r.
In some embodiments, the microcarrier is made af 100%.cellulose.
In- one embodir'nent, differentiated cells may be obtained from stem cells in vitro by a method comprising the steps of: (a) growing stem cells adherent to mir,rocqrrierq in a cuature medium; (b). transferring the microcar'riers from one culture mediUm to another; (c)- optionally repeoting step (b) as reqqirod; ahd (d) qqtaining the-Oifferentiated cdls attac:hed to the microcarrier, The stem cells or partially differeritiated cells may be exposed to a potential r-podulator- whilst still attached to said microcarrier. The invention thus provides a rnethod for using these cells in HTS while still attachecl to the niicroc-~Ir'rier, such as carrying out cel~ transfer steps by Using robotics. The diffe'rentiated cells may be isolated by enzymatic detachment from the microcarrier. The differentiated cells may be isolated by digestion of the microcerrier.
Thp method,L, of the invention may 'he practised using more than 50g dry woight of microcarrier.
Pluripotent stem cells may be grown in vitro by a method corriprising the steps of: (a) seeding said cells on microcarriers; and'-(b) propagating the cells while at'tached__to the carriers.
I
Defiermiination of the identity or cell culture histM of a cell unit Wlien handling large numbers of cell units, their ideptity and/or cell cult6re history (for expmple the chronology ahd the exact nature of a eries of ci~lture conoitions that any one group or unit may have been exposed to) ban beoome; coqfus~ed. Fer instance, the split-pool protocol of cell culture necessarily involves mixing ceil units in each r6und, making it difficult to follow individual uriit$. Qetermiging the cell culture history oF a cell unit in a mixture of cell units which have been subjected to mul-iiple culture conditions is sometimes referred to as 'deoonvolutpan' of thP cell culture history. One method of doing this is to label cbll units and it is therefore advahtageous to label the cell units. Labelling may be perforrned at.the beginriing of pn experiment, or during each round of an experiment ar1o may invol~.e a unioue label. (which may or may not be modified in the course of an experiment) or a series -of labels which comprise a unique aggregate. Similarly, r'eadind of the labei(s) ma~
take place during each round or simply at the end of the Oxperiment. In one p,mbodiment, unique Iabels such as RFID labels are read durin~ each rpund, whareas labels_added serially at each round are read at the end of an ekperime'rjt.
Labelling of cell units may be achieved by a variety of nieans, fQe inst~nce labelling -either, the cells themselves, or any material to which the cells are attached or otherwise .assbciated with. Any of the chemical eind npn-chemical methods qsed to encode synthetic combinatorial libraries can be adapted for -this purpos~ and some of these are described in Methods in Enzymology Vol 267 (1996), 'Cambibatorial Chremistry', John N. Abelson (Editor); Combinatorial Chemistry,s Oxford Uiiiversity Press (2000), Wicham Fenniri (Editor); K. Braeckmans et al., 'Scanriing ihe cbde', Modern Drug Discovery (Feb. 2003); K. Braeckmaris et al,s 'Enc6ding microcarriers:
Present alnd Future Technologies'; Nature Review;s Drpg Discoveryf yol. 1, p, 456 (2002) all of which are herein incorporated by reference. Some examples of labelling tnethods follow.
One method of labelling cell -units involves associatino cell units with a-tag that beeomes sequEhtially modified -as it is placed in different culture donditions. This may involve for instahce the addition or subtraction of further units to the tsg such -th-at its stereochemistry, sequence or mass is altered; or the alteration of electronic Memory as in read=write RF trans-nonders=(see below).
Another method of labelling cell units -involves sequentialiy assi?ciating uniz}pe tags with the cell units whenever they are cultured under dafferent conditioris, sych that subsequeht detection and identification of the tags provides for an unambiguous record of the chronology and identity of the cell culture codiiitions to ~hich the c;ell unit has been exposed. Tags can be taken up by cells, or dttached to the cell surface ~y adsorption, or a suitable ligand pr antibody, or cotijugated to a cell-associated ftii-trix stach as ~ carrier by adsorption, colloidal forces o~ a variety of Iinkeiges such as covalent linltage or rlon-covalent linkage, e.g. biotin-streptavidin linkage. For iristahce, one simple tag that can be introduced to cell's qr attached to a matrix associated with cells is an oligonucleotide of definad length ar,id/or s6qdence.
Oligonucleotides may comprise any class of nucleic acid (e.g. RNA, DNA, PNA, linear, circular pr viral) arsd may contain specific sequences f6r amplifiication (e.g.
primer sequences for PCR) or labels for detection (d.g. fluorophores or quenchers, or isotopic t,ags). T'he detection of these. may be direct, for instapce by seduehcing the oligos or by hybridising them to complementary sequences (e.g. on an -arrihy or chip), or indirect as by monitoring an oligonucleotide-encoded gene prpddrt, or the iriterference of the nucleotide with a cellular activity (e+g; aiitisense inhibition of a particuiar Oerie). An-advantageous-method_of amprifying rtucle'c acids is by rollin j circle amplification (RCA; 2002, V. Dernidov, Expert Rev. flifol, Diagn. 2(6), p.89-95) where nucleic acid-tags can comprise RCA templates, eiongetipn primers, or struts that aid the circularizatio.--T of rrrirnicircle templates).
Any molecular or macromoiecular tag can be used so long as it can be aetecteo, including peptide tags, coio.ured or fluorescept compoUnds, secondary dmines-, halocarbons, mixtures of stabteisotapes etc. Tags may attach to i:ell unIts dirertly or Via an interrnediary, for instance an antibody reisEd against a comporierit of the cell unit, or via an interacting pair such as biotin-steptavidin. In adifition tags can be protected against degradation by the components of the cell culture, for example by chemical br other modification or by encapsulation. Encapsulation of tags can take place in many different media, for example in beads rnany types of which ar'e available from suppliers stach as Bangs Laboratories Inc. .(Fishers IN, -OSA);
and encapsulati-an may be used to standardise tag -dosage in addition to providing cohiiaonents for tag ar'riplification- -and/or detection (for exarihpie -by prqviding PCR
primers for ute with a DNA tag). One method of labelling cell ianits employs fluorescant beads-such as those-manufactured by Luminex Ccirporatlon (Austin, TX, USA)-. The Luminex system comprises. polystyrene beads which may or may not be externally derivatised (e.g; With avidin or aniba-dy) and are intemally dyed -with differing-ratios of two speatrally distin.ct fiuorophor-es,- and a rezrder=wftiGh is capable of chafacteriSing the spectral signature of each bead. A forther method employs beads soch as those manufactured- by Bangs L,aboratories Ino. (Fishers IN, USA).
The Bangs system comprises bead sets which can be distinguisH I ed based on differing sizes (e.g. bead sets of 4.411tn and 5.5 m oiameter). Beads within eqch set can. be fOrthermore distinguished from each other based ori dif'fering fiuprescence intensity owing to differential loading with a single fluoroscent tlye. It is possible to use many different dyes with different absorption or emission characteristics, Which can be inte~n6lly loaded or attached externally to carriers by a rimultiplicity pf ineqns. It Is furthermore possible to use 'quantum dots' to obtain a very' high number of different fluorescent labels which can be read conveniently.
Cell groWth substrates such as those described in connectioh with .forrr~ing cell units can be derivatised or coated with substances that facilitato tagging an-d do-not interfere with cell growth. One method of derivatisjng carriers is to modify them covalently or non-covalently with biotin, to which atag ean bo att~c;hed via -streptavidin or avidin. In general it will be--important to uqe a-t;~g that-wil.l -rot itself induce- a eellular effect (i.e. an inert tag), and that can be djstinguished from i niolecules prespnt in cell units or the culture medial, and -that can be attitch.ed-to its target and suk'rsequently deteeted in the background of such-r'rielecules.
Tb.fjacilitate detection, it may be.-adVahtageous to selectively elute tags~ from cell.units -orto strip off the cdlls from cell units usirig selective conditions. Mpre complicated molecular Jag.ging strategies can also be ehvisaged, including the strqt0-gy of 'bin~,ry -emcqding' where information is recorded by a set of binary codes assigned to a set of rrio(ecylar tags and their mixtures.
IDetection of tags can be accomplished by a variety of fnethods ftimiliar to thos,e skiiied in the art. Methods include mass spectrometry, nuciepr magnetic resonance, se~uenping, hybridisation, antigen detection, electraphoresis, sp ectroscppy, microscopy, image analysis, fluQrescence detection, etc.
Of particular irrterest are- labelling or encoding strategies in wr'ztch-labelling is carried out only c-nce-or where labelling and/or detection -are non-physical and-th.erefore non-invasive. Rarii6frequency Identification (RFID) is-an exarrple-of a system exhibiting these properties. RFID empioys transponders (RF tags), antenr}ae and reatlers.
An RF tag is a-small electr-onic-circuit, usually encased. iH glass or plasiic, which in its -simplest form provides access to- a unique identificatioii codo, that may ~e 'read', ~n-ithout contact or line of sight, by suitable electronics. Tags may alsc store information generated by the user, agaih without contact or line of sight.
A'reader' is an -eleotronic unit that transfers information to and from one or more tags (it should be noted that'the term reader is used interchangeably to mean both a read ohly and iead/write unit). The size and features of a reader may vpry considerably, and jt may operate ir1 isolation, or be cctnnected to a reinote compVater 'sydtem. An ohtenna is used te transtnit information from a reader to a tag, and to receive inforrnation sent by an RF tag. The size and format of an antenna will reflect the specilic appllcation, and may range from a srnall circular coil to large planer structures. An ~FID
system may operate in isolation, or be connected to a remote computer f& more comprehensi~e interpretation and manipulation of idebtification and a isociated data cterived frorri a tag. One RFID strategy used in combinatorial chemistry is described in Nicolaou et al, (1995, AngAw Cherr1 Intl Ed Engi, vol. 341 p. 22$9) arid cpmprjses:
(i) a porous enclosure -containing a synthesis substrate and the sei-riiconductor tag;
(ii) the solid phase syhthesis resin; (iii) a glass- encased Single or Multiple Addressable IRadiofrequency Tag semiconductor unit capable of receiviho, storing anct emitting -radiofrequency tignals. A similar- device could be adaptec!-to g'inr and following cell units simply by replacing the solid phase synthesis resin with tissue culture rraicrocarriers or- suitabi'e-cell units. Mor-e variations of th-is can be envisagod including but not limited to (coated -or uncoated) RF tags bn which cells. are g~owr-r dir-ectly, or RF tags implanted into cell units or organisms;
Thus tags do not necessarily have to be distinguished by their chemical or mol6cular structur=e in the first instance. Multiple variations of the rion-che'nical taoglng strategy can be devieed to determine the identity of a given cell unit In a mitcture or of deducing the identity of the different cell units that cotlnprise 6 mixture.
F6r i~stance optical or visual methods of tagging have been described i~here dlfferent sl?aped objects, graphically encoded objects or different colours denpte the identity df a sample (for example see 1998, Guiles et al, Angew. Qhem. Iritl Ed Engl, v6l.
37, p926; Liaminex Corp, Austin TX, USA; BD Bioscieqces; Merrtopead Technolo'
ie.s, Ghent, Belgium), or wh-ere a patterrl or bar code is etched onto a subs'tra}e-such as a ceromic- bar or nanowire and recognised using pattern recogriition technplogy (for exer-npl.e see 1-997, Xiao et al, Angew. Chem. Intl Ed Engl, vol 8E, p780;
SmartBead Technologies, Babrahajn, UK; Oxonica Ltd', Kidlington, UK).
A further methpd of tracking or.labelling cell- units is to encode thbir ideptity spatially, i.e. by their position in space. In this method-different cOll unjts -are segregated in defined relative positions, and these positions denote or encode the identity of the units. For instance, cell uriits may be cultured in an array, whereby the identjty and/or culture history of each unit is known and is associated to a particular potition in the array. In their simplest forms such arrays can comprise collecticins qf tissue 90lture flasks, wells of a multi-well plate, or locations on a glass slide or other surface.
Examples of positional encoding strategies can be foUnd in GeysEn et 41.
(1984, Proc Natl Acad Sci USA vol. 81, p. 3998-4002), Fodor et al. (1991, Science vol.
251, p.
767-773), Liauddin and Sabatini (2001, Nature, Vol. 411, p. 107-110), and Wu et al.
(2002, Trerids Cell Biol. Vol. 12(10), p.485-8).
The Inventiop has many facets, each of which may have many forms that may be combined to form numerous permutations of the invention. It WiIl be appareht that it is nDt nece;;sar~ to label all of the cell Units in order to be a6 to deduce infori-netion ~bout tho oOtoomes resuitent from a combination bf cell culture protocols.
Thus v~ithout Iebelling of cell units it would still be possible to a'spay large co#inetions of cell culi'<ure conditions accbrding tq the invention, and to determine whethor oiid or rnofe of these was capable of resulting in a particular celluic-rr effer.t.
Hoyhrev-er, in one embodiment, cell units are labelled. Labelling of a cell unit alla'yys, the deri, I vation of useful itiformation from the experirhent regarding the outbome of the p~rticular conditions sampled by the labelled cell unit, as oppqsed to all the ell upits.
AIternatively it ls sometimes advantageous to. label on-e or a few grou.p(s) of cell -units which haere all been exposed to a certain culture protobol, far ihstance a group of cell linits which have been segregated into the same medium dur-ing a particular split or pool step. It will also be apparent that labelling certain cell units allows pna to infer the idedtity of other (perhaps unlabelled) cell units.
Similarly, it will be clear that performing cell culture experirnents iri in~6-h various conditiqrVs are omitted can give infqrmation regarding the utility of those cbnditions with respect to a particular experimental outcome. It v,rould therefore bp ppssibie to (2-valuate each of the conditions sampled in a manher acpordibg to the icveritioii by repeatina. the experiment a number of times, each time on~ittihg a dif6re9t set of conditions.
Split=split cell culture steps can also be used to deterr)ine the effect r;f-e paqicuiar set of conditions on sxperimental butcome. In effect split=spiit ~teps result ii) the format~on- of particuiar lineages of cell units which have eech bee~--exposed to a.
unique -cell culture coriditions at the time of br.anchir.g-. B~ s-ludying the d'ifferent lineages it is possible to determine the utility of the tissue culture cdndltitjns studied at the branching point, with respect.to a particular exoerimOntal outcome:
Haematop6ietlc cell In a further aspect, there is provided a method for proodcing a haematopoietic cell from a s,tetn cell - such as an embryonic stem cell - in vitro gomprising exposing said stem coll to one or more, preferably, two or rnore, reaction conditions, wherein said ~eaction conditions comprise incubating said stem cell vvith: (a) retinoic acid, diniethyl'sulphoxide (DMSO) and/or stem cell factor (SCF); and (b) insulin, steni cell factor (SCF), TGF beta 1, BMP2, BMP4 and/or TPO; and (c) IL-3, IL-6, TP_O, EPO
atid/or M-CSF.
The stem cell may be seeded on a microcarrier - such as a gelatih microc,arrier.
Typically, the stem cells are contained a medium - such as On IMDM basW
(nediqM
or a Streamline Haematopoietic Expansion Medium.
Si-uitably, in step (a) -retlnoic acid or dimethylsulp.hoXide (DMSQ) or stem cell factor (~3CF) bre usbd; suitably in step- (b) insulin alone is used. Suitably, an' step (b) SCF, TGF bete 1, 8MP2 and TPO is used. Suitably, in step (c) IL-3 and IL-0 ~re us~d and optionally TF'O, EPO and/ar M-CS~F are used.
Typically, step (a) is perfoi-med on day 1. Typically, step (b) is perfc-hmec) arl day 4.
Typically; step (c) is performed on day 6.
'n one specpfic embodiment, the conditions are to incubate thE ptem celi with retiiioic acid, dimethylsulphoxide (DMSO), or stem cell factor (SCF); then incul)ate the stem cell with insulin, stem cell factor (SCF), TGF beta 1, E3MP2 or 4 and TPO or incubate the stem- cell- with insulin alone and/or a combination of SCF, TCF beta 1, PYP,-? arrd TPO; and then incubate the stem cell with IL-3, IL-6, and-optiortally TPO, FF'O and/or M-t-SF.
in another specific embodiment, the conditions are on day 1incubate the st'et'n-ceA
with retinoic kid or dimethylsulphoxide (DMSO), or stem cell -Facior (SGi=);
on day 4 incubate the stem cell with stem cell factor (SCF), TGF beta 1, BiqP2, B~1i'IP~4 and TP(~ or incubate the stem cell with insulin alone and/or a combinatirjn of ~ICF, TGF'
beta 1, B02 and TPO; on day 6 inCubate the stem cell with IL-3, IL-6, 1nd optio'nally TPO, EPO and/or M-CSF.
Further aspbcts Further aspects of this invention are presented in the accompanying paragtapMs:
1. A method for identifying a potential modulatQr of a cell signallino pai<hway, comprising the steps bf:
(a) providing a cell of a 1=irst cell type wherein said first bell type may be differentiated to a second cell type by sequentially exposing said first_cell type to one or more reaction ponditions;
(b) adding to or replacinq at least one of said one or rnore reaclion conditions with exposure to one or more different reaction conditions comprising saqcd potential modulator; and (c) monitoring. the differentiation of the first cell type tb determine formation of the second cell type.
2. A method for identifying a potential medulatpr of a-cell signalliiig pathway, cumprisinq the steps of:
(a) providing a cell of a first cell type, wherein said fit=sf cell type is qbtbined from an embry;o or foetus and may be differentiated to a seiz:ond cell typle;
(b) ojStipnaQy amplifying the sajd .first cell type;
(c) further differentiating the said first cell type by sequF I ;ntially expo6ing ~aid first cell type to one or more reaction oonditions;
(d) e>cposing the fir'st cell type to one or more diffo.rent reactian conditions corripriping said potential modylator; and (e) monitoring the differ-e'ntiation of the first cell type cel.I to determine formation of -the secon-d cell type.
3. A rriethod according to paragraph 1 or 2, wherein- the reaction condjtians are the culturb conditions-to which cells are exposed.
4. A method according to paragraph 2 or 3, wherein the first cell ty-pe is a-cell which has been an-ested- along a differentiation pathvvely -between a stern cell and a differentiated cell type.
5. A method according to paragraph I or 3 wherein the cell type is a prirriary cell, cell line or iumour derived cell line.
G. A niethod according to paragraph 4 or 5 wherein the tissue of origin c;rf the cell type i~ selected froin a group consisting of brain, heart, liver, lung, kidn4y, ~skiii, hair, eye, tooth, pancreas, muscle, bone and vasculatu're.
6 PCT/GB2006/004483
7. A method according to any preceding paragraph, wherein the poten)ial modulator is an inhibitdr of a cell signalling pathway.
8. A method* adcor.ding to paragraphs 1 to 6, wherein the potential rnodulator is a promoter of a ceii sig nalling pathway.
9. A mdthod according to pa'ragraphs 3 to 8 wherein the culture conditions required --to differentiate-a cell-ty_pe ?re first identified, in a method-corrjprising the-ste fis of:
(a) providing a first- set of groups of cell units each comprising one or more cells, and exposing-said groups to desired culture conditions;
(b) subdivtdirrg clne or more of said groups to create d further set of ~roups of pell units;
(c) exposing said further groups to further desired culture conditions;
(d) optionally, repeatirig steps (b)-(c) iteratively, as. requpred; and (~) assessing-the effeot on a given cell unit of toe culture conditieins. to which i't has been exppsed.
10. A method according to paragraphs 3 to 8 wherein ttie culture conditions required to partially or fully differentiate a cell type are first itlentified jn a methbd comprising the steps of:
(a) provioino a first-set of groups of cell units each corn f#rising one or rnore cells., an-d expoSing said groups to desired culture_conditions;
(b) pobiing two or more of said groups to form at'ieast one second pool;
(c)- subdiyiding. the second pool to create a further set of grbups of cetl upits;
(d) exppsing said further groups to desired culture-conditioiis;
(e) Optionally, 'repeating steps (b) - (d) iteratively as requi'red; and (f) assessing_ the effect on a given cell unit of the-culture coriditions to Vvhich. it has been expQsod.
11. A rraethod according to paragraph 9 or 10 wherein cell units that are Partially differentiated are then isolated and used in the method of any of clairns I to 8.
12. Use of a pattially differentiated cell type identified in parayraph 9 or ~10 ih the method of any precedir9g claim.
13. A method according to paragraphs 9 tb 12 wherein cell Ljnits are labelled and the label(s) reflect(s) the culture conditions to which the cell unit has been exposdd.
14. A method according to paragraph 13, wherein the label Is spa~ially.encoded.
15. A method according to paragraph 13 or 14, wherein the la6el is selected from the group consisting of a virus, an oligonucieotide, a ppptide, a fluoresc;ent compourid; a secondary amine, a halocarbon, a mixture of stable i6otopes, a bar code, ari optical tag, a bead, a quantum dot and a radipfrequency encdding tag.
16. A method according to paragraph 1-5 wherein- two or more label's .arol selECted and used in comb.ination to label a cell-unit.
17. A method -according to paragraphs 9 to 16, wherein th-e cells are cultured in cell units, each pell unit comprising one or more cells.
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18. A method acpording to paragraph 9 to 16, wherein the cell units are single cells.
19. A rriethod according to paragraph 9 to 17, whoreirl each cell unit comprisss one or rriore ealls adherent to or bounded by a solid substrate.
20. A method aceording to paragraph 19, wherein the solid substrate is a microdarrier or bead.
21, A-metrod ac.cording to paragraph 19; wher+ein thb solid sUbstrate is -a weli or medium-permeable barrier.
22. Amothc?o .according to paragraphs 9lo 21, wherein the- culture conditions are media-to which the cell is-exposed.
23: A method accordirlg to paragraph 22, wherein the media contain one o' moi'e specific agents which ihfluence a cellular process,
24. A rnethQd according to parag'raphs 3 to 23, wherein the cell culture conditions coinpris~ 'csulturing at qne or more specific temperatures or partial pressu'res of oxygen or carbon dioxide.
25. A rriethod according to paragraphs 3 to 24, wherein the cell culturc conditions comprise culturing on one or more specific substrates.
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26. A method according to any preceding paragraph wherEin a firM c'eli is differontiated to a second cell type by modulating cell sigpalling and/or the expression of one or more gene's ih the cell.
27. A method according to paragraph 26, wherein modulation of gene exp~ession ih the cell comprises transfection of said one or mqre genes into the cell.
28. A method according to paragraph 26, wherein modylation of glene expression comprises the exogenous-adn-rinistration of a gEne-produc{.
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29. A method according to any preceding paragraptl wherein the di .ffer-ehtiation of the cell is moriitored by observing the phenotype of the cell or by detecting tr'ie modulation of expression of one or more genes in a cell, there'by de.terrninin'g the state of differentiation of said cell.
30. A rriethod Occordirig to paragraph 29, wherein the modulation of e~b'ression of one or rnore reporter genes is observed wherein the repor(er gene(s) respond(s) to one or more differentiation states of said cell.
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31. A method according to paragraph 29 or 30 wherein the expression of genes involved is monitor-ed qn a gene phip.
32. A method according io- paragraph 29, wherein said one- or ~nore gEne.s encode a-marker.
33. A method according- to paragraph 32, wherein said rparker may- be detected by an iir-.intirroassay.
34. A njethod according to any preceding- method poi~agraph vykerein the differentiation of a cell is monitored by loss of proiii=er~11tive ability.
35. A methbd according to paragraphs 4 or 5 wherein stem, cells or cells that ~ave been detived from stem cells in vitro, are cultufod by a method comprisi-tig t~e steps of:
a) (:pmbining qne or more a.dtures of cells grown under difforbnt conditions;
and b) culturing the cells,
36. A method apcording to paragraph 35, wherein said stem cells are subiected tp at least one 6hange of culture conditions.
07, A method according to paragraph 36, wherein said change of cuiilrre conditions icomprises a change of inedium.
38. A metFrod according to paragraph 4 wherein diffdrentiaj6,-d ceils fiave 4:)eeii obtained from steM cells in vitrb. by a method comprisihq the~~,~teps of:
, (a) Growing stem cells adherent to microcarriers in a culture rryeoium;
(b) Transferring the microcarriers from one .cUlture meoum to another;
(c) Optionally repeating step (b) as required; and (d) Qbtaining tfre differeptiated cells attached to the microcarrier.
39. A method according to paragraph 38, wherein stem cells oi' partially differentiated cells are exposed to said potential modulator whilst still attached to said microcarriei=.
40. A method abcording to paragraph 38, wherein the differentiated cells are isdlated by enzymatic detachment from the microcarrier.
41. A m~.thdd ac-cor-ding to paragraphS 38, 3P or-40 whbrein the process is scdled up such that-dt-least 50g (dry weight) of-microcarrier is-employed.
42. A method-according to paragraph 38 or 41, whereirj the differentiqted -col4s are isolated by digestion of-the microcarrier.
43. The mettlpd according to paragraph 4 wherein pluripotent stem cplls have been grown in vitro by a method comprising the steps of:
(a) seeding said cells on microcarriers; and (b) propagatirig the cells while attached to the carriers.
44. A method according to any fireceding paragraph In which the potential modulator comprises an organic or inorganic small molecule, a natural or Pe'rivatised carbohydrate, protein, polypeptide, peptide, glycop'rotein, nucleic acid, DNA, RNA, oliponucleotide or protein-nucleiq acid (PNA).
45. A method according to any preceding paragraph wherein the potent~al modulator is obtained from.a library of small molecules with drug like properties.
46. Use of a compound library to identify a potential modulator according to any i precedirig paragraph.
47. A pharmaceutical composition comprising a mbdulator ideptified acc6rding_to any preeedirrg paragraph together with a pharmaceutically accEiptable carTiar;
oiluent or expient.
48. Use of a'rrrodulator identified in any preceding paragraph in the -manufacture pf 'a medicament for treatment of a disease.
49. Use of a modulator according to paragraph 48 wher'ein the trealmen-~ is a cell replacement therapy.
50. A partially dlfferentiated cell, which has beqn differentiated in vitro froni a stem cell and arrested along a differentiatjon pathway between h st-om cell and a differentiated cell type.
The invention will now be further described by way.of an Exaniple, whiph is meant to serve to assist one of ordinary skill in :the art in -carrying out the invention. and is not intended in any way to limit the scope of the invention.
Examole A screen was perforrrmed- to identify fegenerative drugs capable of stin'iulating the .myeloid Jineages of the haematopoietic system. The screqn was perfotmed by using a series of culture steps to differentiqte mouse erpbryo# sterrr cells (mESC;',) seeded on microparribrs into progenitors of the myeloid lineages, and su-bsequently 'subjecting these to culture conditions in the presence or absence of th~, lineage specific haematopoietic groWth factors TPO, IVt-CSF and IL-5, or the control compound Vitamin C which is reported tb affect cell suNival but is not haematopoietic. growth factor. After 48 hours the effect on mydloid cell Jifferentiation was assessed using the appearande of macropha(ges as a surrogate assay. This ~crHeii identified TPO and M-CSF es haematopoietic regpherafive drugs capaple of stimulating ttie myeloid lineage.
Materials arid methods Reagents murine s~em-cell factor (SCF) (R&D Systems) murine thmmbopoietin (TPO) (R&D Systems) human erythropoietin (EPO) (R&D Sy.stems) -human interleukin 6 (1L-6) (R&D Systems) human Iransforming growth_factor R1 (TGFR-1) (R&D Systeins) murine macrophage colony stimulating factpr (M-CSF) (R&D Systems) murine interieukin 3 (IL-3) (R&D Systems) retinoic acid (Sigma) human bonp morphogenetic protein 2 (BMP2) (R&D Syst6jis) mouse iritprieukin 5 (IL-5) ((R&D Systems) Ascorbic acid (Vitamin C) (Sign'ia) Microcalture of mESC
Q ulti5pher-G microcarriers (Percell Biolyti-ca AB-) were hydrate.d pyid ste'rilizeo acbordi~ng to the manufqcturer's recor-nmendations.
D3 ES cell (ATTC no. CkL-1934) were grown on gelatine-coated pla.~fir in KG-DMEM cbntaining 15%- knock-out-serum. replacement (KO~R), 1%-non-essential_ amino acids- (NEAA),.1 % Glutamax, .0:5% peniciliin/streptomycin, 0.1_mM ~--mercaptoethanol ((i-ME; Sigma) and 1.000U/ml Leukemia Inhibitory Factor (LIF;
Chemicon); all from Invitrogen unless indicated otherwise.
On the day preceding day 1 of the experiment, approximately 1.5 x 104 biotinylated micocarriers equilibreted in medium A(IMC1M (Gibco), '1~ io KGs~, 1 /Q NEAA, b.5%
pen/strep, U.1rriM R-ME, 1000U/ml LIF and 1.5x1Q'4MI 1-thioglycerol (IVI'('G;
igma)) were added to 100mi of medium A containing approximat~ly 4.5 x 106 ES cells, split into three equal aliquots placed in wells of a 100mm square petri dish (Bibby Sterilin) and incubated overnight.
Preparation of progenitors from mESC
Irn order tq preorare myeioid progenitors a two step mOthod wa; emplbyed.
Beads seeded with MESC as described- above were incubated in IMDM containin4y 1a'BM
retinoic acid for 72h. The beads were then washod in PBS and transferrpc;( to ~temiineT' Naematopoietic Expapsion Medium (Sigma) coritaining ~Ong/ml SCF, 2.5ng T'bFp'!-, 5ng/ml BM'2 artd 20nd/ml TPO and-incubatpd for 4~3h:
Cell-ba~ed assay for regenerative drugs Beads ~aaring rnyeioid progenitors prepared as describeei above were rni*ed, washed in PBS and transferred to the Test (arowth Medium (StemiineTM
~Iaematopoietic Expansion Medium (Sigma) suppiemerited with 3Ong/ml IL-3 and 20rig/ml IL-6). Equal aliquots of approximately 100 Oeads were dispensed iijteb the vvells of a 48 -well plate and incubated in the presence or absence of 50 g/~l Vitamin C, 10ng7ml Il,-5, 20ng/ml TPO and 20ng/ml M-CSF. The screen was cartied out in triplicate In wells of separate plates placed in the same incubatpr.
After 48h, 1 m0 of the macrophage assay reagent DQ-ovalbur~iri (Moiecbiar Probes) was-made up in 0.4m1 PBS and-added'to each well at a dilution of 1:1-00.
Folkiwing inc4bation f.or: at least 4h, the Medium was aspirated ant! -riM_piaced with 1~8,53. The -samples-vioere examined on a Nikon TE2000-S inverted epiflubrescen~
microsdope using a-FITC filter set to quahtitate microcarriers bearin-g large, round cells intornally labelled With green fluorescence.
Th6- results of the screen are reported- as- the aderage nurpber (per cent-) pf -rnicr-ocarriers that--were decorated with macrophage. -In Test Growth i~iediUrn Faorie (i.e. in thP absence of any test reagents) the averade conversion to njacrophago was 1%, which was taken as the basal level of conversion qwing to spontaneous differentiation. When tho negative control oompound Vitamin C Was used as the test i'eagent the average conversion to macrophage was 0%; i.e. below ihe basal level, indicatin0 it had no influence on differentiation. When the haematqbdietic growth factor IL-5 was used as the test reagent the average cohyersion to macrophage was 0%, i.e. al'so below the basal level, indicating it had no influence on differenliation of myeloid cells in this assay. IL-5 is known to influence the lymphoid haerriatoiloietic lineage but has no notable effects on the hiyeloid branch. However, when either TPO
or M-CSF was used as the test reagent, the average. conversion to macrciphage was increased to 6%, repre.'senting a significant increase over backgroL,Ind.
This mESC-based assay is. therefore c-apabie of identifying reagents infhich aet to differentiate myelbid progenitors and could therefore be used to screen libraries of chemical compounds to ide,ntify novel regenerative drug$.
All publication~ mentioned in the above specification are herein jncorporated by reference. Various modificatioris and variations of the described rnethods and system of the invention will be apparent to those skilled in the srt without departing from the scope dnd spirit of the invention. Although the in~ention has beleri descrihed in connection with specific embodiments, it should be understood that theF
invention-as claimed should not be unduly limited to such specific embcdiments. Indeed, various riiodifications of the described modes for carrying out the inventiop whioh are qbvious to those skilled- in molecular biology-or related fields-are intended to bp ~rithin the scope of thb following claims.