CA2122138A1 - A method for enriching fetal progenitor cells from maternal blood - Google Patents

Abstract

The present invention provides a method for enriching fetal progenitor cells from maternal blood, comprising the steps of (a) incubating a sample of maternal blood with an immobilized ligand capable of binding to fetal progenitor cells under conditions and for a time sufficient to allow specific binding of the ligand to the cells, and (b) removing unbound blood products such that the fetal progenitor cells are enriched.

Description

2 1 2 2 1 3 8 Pcr/uss2/o9o24 . " 1 A MEYHOD FO:R ~iNl~G F~ETAL PROGENITOR OELLS
S F~OM ~TERNAL BLOOD

~ss Refer~nce to Rel~ç~li~
This application is a continuation^in-part of pending lLJ.S.
10 Application Serial No. 07/513,057, ~iled April 23, 1990.

Technical Field The invention relates to a method of enriching ~etal progenitor cells from maternal blood.
~a~kgr~und ~the ~nventi~n ~ ppro~imately 5.3 million women become pregl~ant in the IJnited $tates yearly, resulting in 3~8 million del*eries. There are an additional 10 million deliveries in the other af~uent countnes i~ the world. Pre~atal testiIlg is used Ln a ~: 20 subgroup of these women who have a si~ficant risk of ha~ing a fetus with a genetic disorder such as Dowll's Syndrome.
At present, tlhe only way of diagnosirlg fetal disorders is to obtain cells from~ the amniotic iluid (amniocentesis) or the surface of th.e ~etal sac c~orionicvillus) of the mother. Th-~se procedures are exper~iYe and carry a risk; ~ ; 25 of s~ontaIleous abortion of between Y2% to 1%. Because ~ the risk of spontaneQus abor~ioIl, these collection procedures are ~eco~nended only for women who are at a high-~isk of carrying a child with a genetic defect. For example, only women o~er age 35 are advîsed to have the test because the risk ofDown's SyIldrome is higher in that group. Some of these women refuse ~he test 3û beeause of the risk of spontaIleous abortîon. Even though many of these high-nsk women are tested, only a i~action of fetuses with Down's Syndrome are de~ec~ed.
The hîgh-risk women represent such a small po~ion of the women having chîldren that the low-risk population stîll delîvers most of the afflic~ed children. Eîghty percent of those chîldren born wieh Down's Syndrome are from the "l~w-risk,"
35 under 35-year-old group~ This situation is also true of many other genetic de~ects or disorders.

WO 93~08269 2 1 2 ~ 1 ~ 8 PC~r/US92/09024 I~ is therefore desirable to provide a test that wnuld resolve this testing dilemma by providi~g a safe method which could be available to all pregnant women, irrespective of the risk factor aIld without nsk of spon~eous abortion. Although it is kn~ that fe~al cells c~rculate ill t~e blood stream of 5 pregnant women ~see Kulozik and Pawlow~tzl~, "Fetal C,'ells in the Maternal Circula~on: Detection by Direct AF~-Immunofluorescence," Human Genetics 62:221-224 (198~)), fetal cells ~re presen~ in such low concentrations that the procedures necessary to isolate them are extremely difficult and time-consuming;For example, ~erzenberg ~t a~, "Fetal Cells in the Blood of Pregnant Women:
10 Detection and Enric~ment by F~uorescence-Activated Cell Sorting," Proc. Nat~
Aca~ Sci.9 USA 76:1453-1455 (1979), used a Fluorescence-Activated Cell Sorter (FACS) in order to detect fetal cells in maternal blood. The procedure used, however, is not adaptable to routine clinical testing procedures, in part, due to the great expense and expertise required to run a FACS machine. The method of 15 Hertzenberg et a~ is also defic~ent ~or routine clinical testing bec~use it requires determinatioll of ~ ~pes. Bianchi et a~ "Direct Hybndization to DNA From Small Numbers of Flow-Sorted Nucleated Newbom Cells," Cytometry 8:197-202 (1987~ also used a FACS machine to detect nucleated cells, althvugb the blood sample was not obtai~ed ~om the mother but from the newborn's umbilical cord.
Tbe prese~t invention provides a metbod for enriching fetal cglls that ~vercomes these disadvantages, and furtber provides other related advalltages.

Sl~mmaly ~f ~e Invention : 25 T~e preseIlt inYen~ion is also directed ~oward methods for enriching feta~ nudeated~ e~hroid cells from maternal blood. Within one aspect of the present inven~ion, such a metXod compAses the s~eps of (a) incuba~dng a sample of matemal blood with an immobilized ligand capable of specifically bi~ding ~o fetal ~, nucleated, e~hroid cells lmder conditions and for a time suf~c~ent to allow 30 specific bindillg of the ligalld to the cells, (b~ remo~g unbound blood products, d (c) ~cubating the bouIId cells in the presence of erythropoietin such that thefetal cells are prefere~ially enrichçd. Withill orle embodiment of this aspect of the inventioD, the immobilized ligand is an immobili~ed antibody.
Within another aspect of the invention, a method is pr~vided for

3~ enriching ~etal nucleated, erythroid cells from maternal blood, compnsing thesteps of: in~bating a sample of maternal blood with a first nnember chemically li~ced to a ligand capable of speci~ically binding ~o fetal nucleated, e~ythroid cells WO ~3/08269 2 ~ Pcr/uss2/o~û24 under conditions and for a time sufficient to allow specific binding of the ligand to the cells; adsorbing the cells to an immobilized second member, tbe second member beL11g capable of binding to the first member with an affinit~ constant of greater thaIl about 108 M-l; remo~ing unbound blood produc~s; and incubating 5 the bound cells ~n the presence of erythropoietin such that the fetal cells are preferentially enriched. Suitable first member~second member binding pairs include biotin-~vidin~ biotin-streptavidin, biocytin-avidin, bio~rtin-streptavidin, ~:methotrexate-dihydrofolate reductase, 5-fluorouracil-thimydylate synthetase, and riboflavin-riboflavin binding protein. Within one embodiment of this aspect of the 10 invention, the first member which is chemically linked to a ligand is a biotinylated antibody and the i~obilized second member is immobilized avidin.
~:Within another aspect of the present invention, the method comprises the steps of: ~ncubating a sample of rnaternal blood with a first ligand capable of specifically binding to fetal nucleated, eIythroid cells under conditions 15 a~d for a time suffi~ient to allow specific binding of the first ligand to the cells, incubating the sample with a first member chemically linked to a second ligand ~;capable of specifically bin~g to the first ligand under conditions and for a time suf~cient to allow the second liga~d:to bind to the first liga~d; adsorbing the cells to aII immobilized second member, the second member capable of binding to the 20 first member with an afEinity coIlstant of greater than about 108 M 1; removing unbound blood~ products; and incubating the bound cells in the presence of erythropoietin such that the fetal cells a~e pre~erentially enriched. Within oneembodiment, the first Iigand is an antibody which specifically binds to fetal nucleated, e~roid cdls. Within preferred embodiments, the first member which 25: is chemically linked to the second ligand is a biotinylated antibody. In such an embod~ent, the immobiliæd second member is immobilized avidin.
As an alternative to the step o~ incubating bound cells in the :~ presence of e~thropoietin, another aspect of the present invention comprises the steps of: (a) incubating the bound cells wi~h ammonia and chloride ions and a 3~ ca~bonic anhydrase i~ibitor under conditions aIId for a time sufficient to allow accumulation of a~nonium ions within the bound cells, and (b) incubating the bound cells containing accumulated ~nmonium ions in the presence o~ ammonia ~:and c~bon dioxide such that selective hemolysis of maternal blood cells occurs.
I~ addition, the present invention may also be perfo~ned usillg a combination of35 both erythropoietin enrichment and the method described above wherein the cells are incubated with ammoI~ia and chloride ions and a carbonic anhydrase inhibitoruIlder conditions and for a time ~u~Scient to allow accumulation of ammon~um Wo 93~8269 Pcr/uS92/09024 212~138 ions urithin tbe bound cells, followed by the incubation of bou~d ceL~ co~taining accumulated ammoDium ioDs in the presence of ammonia and c;~bon dio~ude such th~t selective hemolysis of ma~ernal blood cells occurs. These hvo enrichment methods may be perfolmed sequentially, and in either order.
Within another aspect of the prese~t invention, a method is pr~vided for enriching fetal ~ucleated, er~oid cells from matemal blood, compAsing the steps of: incubating a sample of ma~ernal blood in the presence ofelythropoietin such that the fetal cells are enriched; incubating the enriched cells with an immobilized ligand capable of specifically b;nd~ng to fetal nucleated, 10 elythroid cells under co~ditions and for a time sufficient to allow speci~Sc binding of the ligand to the cells; and removing unbound blood products. Alternatively, within another embodiment of the present invention, the enriched cells may be immobilized by ~ncubating them with a first member chem~cally linked to a ligandcapable of specifically binding to the cells under conditions and for a time 15 sufficient to allow specific binding of the ligand to the cells, and adsorbing the cells to an immobilized second membe-, the second member being capable of billding to the first member with an affinily cor~ t of greater than about 108 M-1 . Wi~n yet ~ ~ther embodiment of the present invention, the enriched cells may be imlmobilized by incubating the enri~ed cells with ~ first ligand 20 capable of speciScally bindLng to the cells under coIlditions aIld for a timesufflcient ts~ allow specific binding of the first ligand to the cells; incllbating the sample with a first member che~cally linked to a second ligand capable of specifically binding to the iirst ligand under conditions and ~or a time sufficient to allow tbe second ~gand to bind to the first ligand; and adsorbing the cells to an 25 immobilized second member, the second member being capable of binding to the fi~st member with ~ ~t~ cons~t of greater than about 10B M-1, Suitable combiIIati~ns of ~rs~ ~nd second ligand are discussed in detail below.
In addition, ~nthin the aspects discussed above, the methods may further comprise ~subsequent to removing the unbound blood products~ the steps 30 of: incuba~ng the bo-~d cells with ammoI~ia and ~hloride ions and a carbol~icanhydrase inhibitor under conditions and for a time suflïcient to allow accumulation of ammonium ions within the bound cells; and incubating the cells containing accumulated ammonium ions in the presence of ammoni~ and c~rbon dioxide, such that selective hemolysis of maternal blood cells occurs.
Within otker aspects of the present inventio~ as an alternative to first incubating a sample of maternal blood in the presence of eIythropoietin, the cells are incubated with ammonia and chloride ions and a carbsnic anhydrase wo 93/08-~69 . Pcr~us92/os~2 iIlhibitor uIIder condi~o~ d for a time su~cicnt to allow accumulalio~ of ammo~um io~s wit~ thc bound c~lls, followet by i~cuba~os of the cells containing accumulatcd ammor~ium ioDs i~ thc prescncc of ammo~a a~d carbo~
dio~ade, such that scle~ve hemolysis of matcr~al blood cells occurs.
S Within the prescnt i~ventia~, a ~ranc~ of ligands may be ut~d, includi~g antl~dics, crythropoie~, a~d ~sfc~ Thc ligand may be immobilized o~ any of a ~rane~r of sa}id supports, such as hollow fibe~s, bcads,magnetic b~ads, platcs, dishes, ~asks, meshcs, scrceDs, solid fibess~ membranes,a~d dipstic3cs.
Withi~ another aspect of thc inYe~on a method for c~nching fc~
proge~i~or cells from maternal blood is pronded compnsing the steps of :~ ~: (a) incubating a samplc of mater~al blood ~th an ~obilized ligand capable of speci~cally binding to fe~al progenitor cells ulldcr condi~ons, and for a ~me sufficierlt to allow specific bindi~g of ~e ligand to t~e cclls, a~d (b) removing 15 uIlbou~d blood products such that thc fe~al progc~itor cells are cnrichcd Within OIlC cmbodimenL prior to the stcp of incuba~ng, red blood cclls are removed ~rommatcrnal blood, for example by n~n~ing the matc~al blood over a ~icoll gradien~
Wi~ anothcr embodimcnt, subsequc~ to the StCp of removing thc unbound blood products, the bound cells are i~albatcd in the presence of erythropoie~
Withi~ anothcr aspect of the prcse~t ~n~en~ion, a method is provided for em;ching fetal progemtor cells ~om matcsnal blood comprising the steps of (a) inalba~g a~ sample of matcrQal blood with a labcled ligand capablc of specifically billdi~g to fetal progeDitor c~lls, under co~di~ons, arld for a ~e sufficient to:allow:speci~ic biIl&g of thc ligand ts the cclls, (b)d~t~c~ing thepr~ence of the~ ligand bou~d~cells~ and (c) ~cpara~ng the liYand bouIld cells ~om the unbou~d c~s,:~such that said fesal proge~itor cells iare cnnchcd. Wi~hini one embadiment, p~ior to the stcp of i~ ba~g, red blood cells aFe removed from mate~al bioo;~ ~y, for cxample, n2n~g tbe matcmal blood ovcr a Ficoll ~ent. Wi~ a~other cmbodime~e, 5UbSalUCalt tO the step of removing the u~bound blood products, the bou~d cells asc iIlcubaud in the prcsence of e~hropoie~ Withi~ the embodi~ients, the labcl is selec~ed from the group ng of ~oresciein-isQthioqanate, phycoe~hrin, rhodamine iso~iocyana~e, : ~ or other such highly ~uoresccnt moleQLes. I~ other e~nbodimcnts of the ~ invention, ~iC ligand ~ ~ibodv such as 1~8.
:~: 35 Withi~ another aspe~t of ~he ~reDi~on. a mcunod is provided for en~iching fe~ progen2tor cells from mate~l blood com~rising, ~hc stcps ~f ~a) incubating a s~nple of materr~ blood with an immobi}iz~d ligand capable ot Wo 93/08269 Pcr/uss2/o9o24 2122138 `~`~

specifically binding to cells other than fetal progeDitor cells, under condiffoIls and for a time sufflcient to allow specific binding of the ligand to said other cells, and ~b) remc~ving the nonbound fetal progenitor cells, such that said fetal progenitor cells are enriched. Within one embodiment, prior to the step of incuba~dng, red S blood cells are removed from maternal blood by, for example, running the maternal blood over a Ficoll gradient. Within a preferred embodiment, the ligandis a~ antibody.
Within another aspect of the present invention a method is provided for typing chromosomes of fetal nucleated erythroid cells, comprising the steps of 10 (a) incubating the fetal nucleated erythroid cells in a media contaiI~ing e~thropoietin under conditions and for a time sufficient to induce metaphase in the cells, (b) f;xing the DNA of the cells~ (c) staining the fixed DNA such thatchromosomes may be obsenred, and (d) examining the stained DNA thereby allowing the ~ping of the chromosomes.
These and other aspects of the present invention will become evident upon reference to the following detailed description.

Detailed Desc~i~tion of the Invention As noted above, within one aspect of the present invention methods 20 are provided for the enric~ment of fetal nucleated, erythroid cells from maternal blood. Maternal blood colltains, among many other ~pes of cells, both adult and fetal nucleated, e~ythroid cells. Througb the efforts of the present invention, fetal nucleated, erythro~id cells~ may be enriched from as few as 1 in 106 in.maternalblood, to an enr~ched concentration of about 1 in 103, and preferably, to about 1 25 in 102. Wi~n the context of the present invention, nucleated el~rthroid cellscontain a nucleus and generally include e~ythroblasts as well as other erythroidprècursor cells.
Maternal blood may be obtained from a pregna~t female using conventional techniques well Lnown in the art. Preferably, peripheral blood is 30 drawII from an e~ily obtainable source such as the antecubital vein ~the arm vein) with conventional venipuncture techniques. Once the maternal blood has been drawn, it may be frozen using conventional techniques, or stored at 4C for a maximum of 4 to 7 days. Various anticoagulants may be added to the blood as ne~essaly, including, among others, ACD, CPDA, EDTA, and Heparin.
The maternal blood is then subjec~ed to a selection method in accordance wi~h ~he present invention, wherein preferentially enriched fetal cells may be obtained urithout the need for further purif;cation, for example, by a WO 93/08269 2 1 2 2 1 3 8 Pcr/vss2/o9o24 Fluorescence-Activated Cell Sorter (FA(~S). In general, ~he methods of tbe presellt invention comprise the steps of: (1) incubating the maternal blood witheither an immobilized ligand, or a ligand which will subsequently be immo~ilized, such that the ligand binds to and hence immobilizes the fetal ~ucleated, erythroid S cells, (2) remo~nng unbound blood products, aIId ~3) preferentially enriching the bound cells for fetal nucleated, erythroid cells. As noted above, these basic steps may be performed in an alternative order, for example, comprising the steps of:
(1)preferentially enriching maternal blood for fetal nucleated, eIythroid cellsj~2) incubating the enriched cells with either an i~nmobilized ligand, or a ligand 10 which will subsequently be immobilized, such that the ligand binds to and hence immobilizes the fetal nucleated, erythroid cells, and (3) removing unbound bloodproducts. The present invention may be perforrned utilizing devices as describedin an application entitled "I~unoselection Device and Method," U.S. Serial No.
07/513,S43, and an application entitled "An Apparatus and Method for Separating 15 Particles Using a Pliable Vessel," U.S. Serial Mo. 07/599,796, both of which are incorporated herein by reference.
In addition, one may utilize an immunoaffinity column as described in pending U.S. Application ~Attorneys Docket No. 200072.407) Serial No.
entitled "Improved ~pparatus and Method for Cell Separation"
20 (hereby incorporated by reference in its entire~). Briefly, within one aspect of this application, a "cell separator" is provided, including a colurnn assembly for separating target cells ~rom a sample fluid, the column assembly including a colu~, a sample fluid supply bag and a fluid collection bag wherein the column is provided for receiving the sample fluid ~om the sample fluid supply bag and for 25 separatillg ~e target cells ~om the sample ~uid and retaining the ta~get cells, and wherein:the ffuid collection bag is provided for receivi~g the ta~get cells after being released ~om the column, said cell separator comprising aIl agitation mea~s for a~tating the contents of the column to assist in releasing the sample cells retained in the column, the agitation mealls being responsive to a drive signal for 30 va~ying amount of agitation of the contents of the column to va~y the ra~e at which he sample cells are released, column sensor means for providing a column signal indicative of the optical densi~ly of ~uid flowing out of the column and into the fluid collection bag, a column valve means response to a column valve control signal for selectively enabling the fluid coming out of the column to flow into the 35 ~luid collection bag, axld a data processor means for controlling the operation of the cell separator, the data processor means being response to the column signalfor providing the drive signal and the column valve con~rol signal to prevent WO 93/0826~ P~r/uss2/~9o24 inadequ~te concentra~ions of the target cells from being collected. One embod~ent of ~is invention is tbe OEPRAT~ I~n' cell sep~ratio~ system which is available from CellPro0 (Bothell, Wash.).
Within one aspect of the present inve~tion, the mate~al blood is S incubated with an immobilized lîgand capable of spe~ifically binding to ~etal nucleated, erythroid cells ullder conditions, and f~r a time sufficient to allowbinding of the cells to the ligand. Generally, incubation of about 15 to 30 ~utes at 4C to 37C is preferred. If the incubation step occurs as the cells are passed.
~ver a column, ~he flow rate should be suf~ciently slow to allow the cells to bind.
10 Pre~rably, the cells should be allowed at }east 15 ~utes in which to traverse the bed of the column.
As noted above, the ligand should be chosen such that it is capable of specifically bLnding fetal mlcleated, elythroid cells. Within the context of the present i~vention, the ligalld is def;ned to be "specifically binding" if it is capable of binding to fetal nucleated, erythroid cells, but not to more than about 10~o of the matemal blood cells. The relative percentage of bound fet~l nucleated, elrythroid cells to other cells m~y be readily de~e~ined by analysis w,ith a specific marker such as Alpha Fetal Protein (AFP). For example, at a~y point within ~e methods discussed below, the relative percentage of fetal cells to rnaternal cells :: 20 may be de~ermined with either glucose o~dase or fluorescein conjugated anti-AFP an~body. See ~ ~ulozilc and I. H. Pawlowitzki, "Fet~l Cells in the Maternal Circulation: Detection by Direct AF~-Immunofluorescence," ~Iuman Genet.
62:221 (1982). This determination is pr~erably performed af~er ~vo Dl more pu~ification steps.
2~ IigaJlds whic~ spe~ifically bind to fetal nucleated, erythroid cells are knouql in the ar~"ncluding e~hropoietill (Amgen, ~ousand Oalcs, Calif.), ~: ~ transfe~i~ (Sigma (:hemieal Co., St. Louis, Mo.) and sele~ed a~tibodies.
~onoclonal antibodies whicb spe~ically recognize Ilucleated erythroid cells are particularly preferred. Monoclonal antibodies to nucleated e~ythroid cells such as 30 allti~ ferrin receptor antibodies may be ob~ained from conYentional suppliers(~ecton Dickinson Immunocytometry Systems~ Mowltain View, Calif.~.
Alternatively, monoclonal anti-erythroid antibodies, such as EP-1, may be produced using techniqlles well kn~ the art. See Yokochi ~t a~, "MoIloclonal Antibodies Detecting Antigenic Determinants With Res~ricted l~xpression On 35 Erythroid Cells: From ~he Erythroid Committed Progenitor Level to the Mature E~ythroblast," Blood 63:1376 ~1984); see also Heddy Zola (ed.), ~Ionoclonal Ar~ibodies: A Manual of Techni~ues, CRC Press, Boca R~on, Fla. ~1987).

WO 93/~8269 Pcr/uss2/o9~24 Briefly7 cells may be generated for ~nization from fetal liver clonal erythroid cultures, and e~iched for progen~tor ~lls. Thu~, ~he poplllati~n o cell~ used for aIltigens aIld for plimary screening may contain ~mmature eIythroblasts, erythrobla~ts of all intermediate degree o matuA~y, and presumably, progenitor cells of BFIJ-E arld CFU-E ~pes. These cells may be used for intravenous immuniza~lio~, followed by removal of the splee~ and fu~ion of the spleen cells with a myeloma line such as NSI, using standard techIIiques. The result~g fused cells, or hybridomas, may then be screened ag~t the above-described cells using conventional techniques. See Yokochi et a~, supra.
The e~tire specifically binding antibody need not be used as the ligand. More specifically, only the binding region of the an~ibody is necessa~y to specifically bLnd fetal nucleated, eIythroid cells. Thus, antibody ~agments such as Fab or F(ab'~2 ~agments may be used within the presen~ inveIltion. Additionally,the binding regions of the specifically binding antibody may be incorporated into a 15 new protein, which may be used as the ligalld. See Reichmann et ~, "Reshaping:~ Human Antibodies For Therapy," Na~ure 332:323-327 ~1988); Verhoeyen et al., "Reshaping Human Antibodies: Grafting an Antilyso~yme Ac~ y," Science 239:1534-1536 (1989); and Robe~s e~ a~, "Generation of an Antibody ~th EnhaIlced ~i~y and Specifici~ ~or its Antigen by Pro~ein l~nginee~g," N~ur~
: 20 328:731-734 (1987).
Within ~he present invention, the ligand is immobilized in order to allow separation: of bound cells from other blood products. Many suitable supports are well l~own in the art alld include, amo~ others, hollow fibers (~con (:orporatiorl, Danvers, Mass.), beads (Polysciences9 Warrington, Penn.), 25 magnetic beads (Robbin Scientific, Mou~tain View, Calif.), plates, dishes andflasks (Corn~g Glass Works, Corning~ N.Y.), meshes (Becton Diclcinson, Mountain View, ~.), screens and solid fibers (see Edelmall et al., U.S. Patent No. 3,843,324; see also Kuroda et al, U.S. Patent No. 4,416,777), membranes ~Mi}lipore Corp., Bed~ord, Mass.~ and dipsticks. A variety of different sources 30 exist for supports other than ~ose designated. Particularly preferred is a support such as Biogel P-60n' (BIC)RAl:), Richmond, C:alif.). Biogel P-60n' is a porous polyacrylamide hydrogel bead. The beads are generally spher~cal, on average about 250 microns in size, and have an average pore size which excludes molecules larger than approximately 60,000 daltons.
3~ A va~ie~ of methods may be used to immobilize the ligand onto a support. For example, a ligand, such as an antibody, may be direct~y coupled to the support by vari~us methods w~ll known in the a~t. 3ee J. K ~marL, Methods In W O 93/08269 PC~r/US92/09024 2 1 2 2 1 3 8 Enymology, Vol. 34,A~qnity Tech~es, EnymePurificatio~ PartB, W. B. Jakoby and M. Wilchek (eds.), Academic Prexs, New York, p. 30 (1974); see also M. Wilchek and W. Bayer, nThe Avidin-Biotin Complex in Bioanalytical Applications,"Ana~t. Biochen~ 171:1-32 (1988). These methods include the use 5 of glutaraldehyde, carbodiimide, carbonyl diimidazole, cyanogen bromide, tosyl~hlo~ide, biotin/avidin, and biotin/streptavidin. Once the ligand has been immobilized onto the support, the maternal blood may be incubated with the immobilized ligazld under conditions, and for a time sufficient to allow binding of the ligand to the cells. Within the context of the present invention, suitable 10 conditions for binding to occur include incubation in a physiological buffer at about 4C to about 37C. Particularly preferred temperatures range ~om about 4C to room temperature. The time of incubation depends on the afflnity and avidi~y of the ligand for the cell, and may be readily determined. Generally, incubation for about 15 minutes to one hour is preferred. Following incubation, 15 unbound blood products may be removed, and fetal cells enriched using the ~; ~ methods describe~ herein.
Within another aspect of the present invention, a sa~nple of maternal blood is incubated under suitable conditions with a ligand which is chemically linked to a first member, and then adsorbed to a second member 20 which is immobilized on a solid support. The first member should be capable of binding to the second member with an aflini~y of greater than about 108 M~1 Many suitable f;rst member-second member binding pairs are well l~own in the art. These include, among others, biotin-avidin, biotin-streptavidin, bio~ytin-andin, biog~tin-streptavidin, methotrexate-dihydrofolate reductase, 25 S-fluorouracil-thimydylate synthetase, riboilavin-riboflavin binding prote;n,antibody-protein A, and antibody-protein G. In a preferred embodiment, the firstmember is biotin and the second member is avidin.
Either member of the above described binding pairs may function as the second member, with the complementa~y member functioning as the first 30 member. Furthermore, combinations of the first member-second member binding pair may be employed. For example, biotin may be linked to the ligand, as well as adsorbed to the suppon. The cell, ligand, biotin-eomplex and biotin, support-complex may then be bound together through an incubation step with andin.
Avidin is multivalent, permitting the ~ormation of a cell, ligand, biotin, avidin, 35 biotin, support-complex which immobili~es the cell.
Within one example of this embodiment, a sample o~ maternal blood is incubated with a biotinylated antibody under conditions and for a time WO 93tn8~69 21 2 2 ~ ~ 8 Pcr/us92/oso24 sufficient to allow binding to occur. 'rbe sample is then incubated wi~h, or passed over, a support which contains immobilized andin. Cells which are coupled to thebiotinylated antibody are adsorbed to the immobilized avidin, thus allowing separation of cells from unb~und blood products. Subsequently, unbound blood S products may be removed, a~ld fetal cells enriched using methods described below.
Within yet a~other aspect of the present invention, a tw~step method is used to immobilize tbe fetal nucleated, erythroid cells. Brie~y, ~ first ligand is incubated with a sample of the maternal blood under suitable conditions as described above. Subsequently, a second ligand which has been chemically 10 linlced to a first member is added. 'rhe second ligand is capable of bind~ng to the first ligand. The cell, first ligand, second ligaIld, first member-complex may then be adsorbed onto an immobilized second member, t~us allowing the separation of cells ~om unbound blood products. Representative examples of the first member-second member binding pair have been discussed above. Representative 15 ~ examples of the first ligand include eIythropoietin, transferrin and selected antibo~es. Once the first !igand has been selected, the second ligand is chosen such that it specifically recogluzes and binds to the hrst ligand. Within a preferred embodimenL the second ligand is an antibody, for example, an anti-e~thropoietin (Teny Fox Laboratory, Vancouver, B.C., Canada), anti-transferrin (Chemicon 20 Intl., Inc., Teme~ula, C~alif.), or anti-immunoglobulin antibody. Anti-immunoglobulin antibodies may be prepared using techniques well hlown in the art, or may be ~ined ~from conventiona~ sources, including, among others, Sigma Chemical Co., St. Louis, Mo., and Becton Dickinson Immunocytometry Systems, M~untain ~riew, Calif.
25 ~ ~ Within a preferred embodiment, the first ligand is an antibody which specifically~recognizes fetal Ilucleated, erythroid cells such as an anti-; transferrin receptor antibody (Becton Dicldnson Immuno~ytometry Systems, Mo~tain View, C~lif.). The antibody is ~ncubated with a sample of matemal ~, blood. A biotinylated anti-immunoglo~ulin alltibody, such as biotirlylated goat 30 anti-mouse lgG ~the second lig~d which is cbemically linked to a first member) is then added and iIlcubated with the sample. The sample is then incubated u ith, or passed over a bed of material which contains the immobilized second member, in this case, immobilized avidin. The cell, antibody, anti-immiunoglobulin antibody, and biotin-eomplex will be adsorbed to the immobilized avidin, thus allowing the35 subsequent removal of unbound blood products.
As noted aboYe, once the cells have been immobiliz~d, unbound blood products may be removed. In one embodiment, the immobilized cells are WO 93/08269 Pcr/us92/09024 j~ ~,, rinsed with a physiological bu~er9 thereby removing the unbound blood products~
Va~ious methods may be used to ~inse the immobilized cells, depending upon the type of support chosen. These methods include, among others: washing or flushing the support; magnetically attracting the support out of solution, followed S by resuspension in a physiological buf~er; and centrifuga~ion followed by resuspension. Various physiological buffers are also well known in the art, including PBS, PBS plus albumin, such as Bovine Serum Albumin ~BSA), normal saline and cell culture rnedi~.
Once unbound blood products bave been removed, bound cells may 10 be preferentially enriched ~r fetal nucleated, e~ythroid cel~s. As noted above, at least hvo alternative methods may be used either separately, or together. Lf thetwo methods are performed together, either method may be performed first.
Within one embodiment the bound cells are cultured under selected culture conditions in the presence of elythropoietin (Amgen, Thousand Oaks, Calif.). SeeEmerson et a~, "Developmental Regulation of Erythropoiesis by Hem~topoietic Growth Factors: Analysis on Populations of BFU~E From Bone Marrow, Peripheral Blood and~ Fetal Liver," Blood 74(1):49-55 ~1989); see also Iinch et a~, ~;: "Studies of Circulating Hemopoietic Progenitor Cells in Huunan Fetal Blood,"
Blood 59(5~:976-979 (1982). Selected culture conditions generally include growth20 in standard cell c~lture ~media, without any other ~ytoldnes other tha :e~hropoietin. This preferentially allows fetal, but not maternal, nucleated erythroid: cells to grow. Pa~ticularly preferred media includes Iscoves' Modified Dulbecco's Medium (Gibco, GraIId Island, N.Y.) containing a final concentration of 20% fetaI bov~ne serum, and 2 U/ml puri~ed unnaly human EPO.
Within the second method, fetal nucleated, exy~roid cells are preferentially nriched based upon tb,eir uptake of ammo~ium ions and the ~;: selective hemolysis of maternal blood cells, or more speciiïcally, of maternal elythroid cells. See genenalb Jacobs and Stewart, '~he Role of Carbonic :~ ., Anhydrase in Certain Io~ic Exchallges Involving the EIythro~yte,".~. Ger~ Physio~
25:539-552 (1942); and: Maren and Wiley, 'Kinetics of Carbonic Anhydrase in Whole Red Cells as Measured by Transfer of Carbon I:)io~de and Ammonia,"
Molecular Pharmacolo~ 6:430440 (1970). Briefly, the cells are incubated in the ~: : presence of ammonia alld chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions 35 within the cells. 'rhis generally takex approximately 5 to 30 minutes. The cells are then incubated in the presence of ammonia and ca~bon dioxide such that selectivehemolysis of maternal blood cells occurs.

WO 93/08269 2 1 2 2 1 3 8 PCI/U~92/09~24 Withi~ the context of the present invention, many compounds may provide sui~able sources of ammonia, shloride io~s and carbon dio~de. For example, sui~ble sources of ammonia include, among others, ammon-a and the ammonium salts. Suitable sources of chloride ions include, among others~ NaCl, S KCl, MgCi2 and CaC12. ~uitable sources ~or carbon dio~nde anclude9 among others, carbon dioxide iII solution, carbonate and bicarbol~ate.
In addition, many carbonic a~hydrase inhibitors are known in the art3 including, among others, most monovalent ~ons such as ~yanide and cyanate, monovalent sulfides, sulfonamides, and acetazolamide. See Lindskog e~ a~, The 10 Er~ymes 5:587 (1971); Ward and C~ull, Arch Biochem. Biophys. 150:436 (1972~;
and Pocker alld Watamori, Biochem. 12:2475 (1973). The carbo~ic anhydrase inhibitors should b~ selected so as to functi~n under physiological conditions.
Preferred carbonic anhydrase inhibitors include sulfanilarI~ide and acetazola~ide.
Bound cells may be released by various methods either subsequent 15 to, or prior to en~ichment. If bound cells are released prior to erllichmen~, they may be subsequentl~ enriched using the above-described methods. Various ~: methods are known L~ the art ~or releasing cells. Within one such method3 cells may be cul~ured with or without 6~ytoldnes. C~tokines, such as II~2, can cause prol~eration of cells, or cha~ges iII the su~ace characteristics of ~e cells, such ;: 20 that the cells or their progeny are released i~om a support. Within another me~hod, cleavage of the li~and or of the cell-ligand bond may release the cell.
Various cleavalble lig~ds and cleaving enzymes are Icnown in the ar~9 including among others, papain alld t~ypsin. Within yet aIlo~er method, the cells may be : : released by mes~aI~ical, ~avitational or electromagnetic forces. A particularly 25 preferred method i~ mechanical agitation, for ~xample, by agitation of the beads thr~ugh pipett~g, stimng9 sh~g, vibration, or sonic~tion.
~ i ~ithin aIlo~her aspect of the present invention, a method is : provided wherein the cells are i~rst en iched, ~ollowed by adso~ption of fetal ;; ~ I ~ucleated, erythroid cells and removal of unbound blood products. Briefly, fetal 30 cells m~y be enriched by hrst incubating materllal blood wi~h a~noI~a and chloride ions, and a carbonic anhydrase inhibitor. After the cells have been incubated under conditions and for a time sufficient to allow accumulation of anLmonium ions, the cells are ~eated with ammonia and carbon di~xide such that ~: selective hemolysis of maternal blood cells occurs. Fetal cells may also be : 35 enriched by incubation u ith e~hropoietin as discussed above, or by a combined treatment using both erythropoietin and the method discussed above wherein $he Wo 93/08269 PCr/US9~/09024 212~ 138 ";

cells are treated with ammonia and carbon dio~de. Furthermore, as discussed above, the ~NO methods may be performed in either order.
The enriched cells remaining after the method(s) described ab~ve may then be ~obilized usi~g any of the above discussed methods, including:
S (1) an ~obilized ligall~ w~ich specifically binds ~o fetal nucleated, e~roid cells rem~ining after enrichme~t, (2) incubatiIlg cells rem~g after enrichment with a hrst member linked to a ligand, the ligand being capable of specifically binding to fetal nucleated, e~ythroid cells, followed by adso~p~Lion of the cells to an.
immobilized second member, the second member being capable of binding to the 10 fîrst member with an ~ity constant of greater than about 108M-1, and (3) incubating c~lls rem~ng after enrichment with a first ligalld, followed by incubation with a second ligaIld which is chemically linked to a first member which is capable of binding to the first ligand under conditions and for a time sufficient to allow the second ligalld ~o bind to the first ligalld, followed by adsorption of the cells to an ~obilized secolld me~ber, the second member being capable of binding to the first member with an affinity eo:nstant of greater than about o~M-l ~: Subsequent ~o ~obilization of the cells~ unbou~d blood products m~y be removed using methods described above. If the cells were initially en~iched using elythropoietin, subsequent to removing unbound blood products, the cells may be treated using the method wherein ammonia and carbon dioxide are used. Similarly, if these cells were initially treated with the method wherein ~onia alld c~bon dio~ide are used, s~sequent to remov~ng unbound blood products, the cells may be treated with eIy~ropoietin.
Fetal cells which haYe been enricbed from maternal blood may be chara~terized by incubating the preferentially enriched ~ells with a marker c~pable of selectively binding to ~etal cells. A marker selectiYely binds to ~etal cells when its presence on the target cells is greater ~han 1~fold more than thequantity on maternal cells. Various markers are knowD in the art, including, for30 example, ~tibodies to Alpha Fetal Protein ("AFP"). See Kulozik et al., "FetalCells in the Maternal Circulation: l~etection by Direct AFP-Immunofluor~ssence," Human Genet. 62:221-224 (1982), or antibodies to antigen "i". See Y.W. Kan et al., "C: oncentration of Fetal Red Blood Cells From a Mvsture of MaterIlal and Fetal Blood by Anti-i Serurn," Blood 43(3):411~15 (1974).
35 Antibodies to the fetal cell marker may be labeled using techniques well known in the a~t and used to detect the presence of fetal cells.

WO 93J08269 2 1 2 2 1 3 8 Pcr/uss2/09024 ~5 Within a prefelred embodimellt of the present ~vention, liga~d is removed from the cell surface through ~he ~ethod described in a related application (U.S. Serial No. 07/513,056) elltitled "Methods for Removing Iigallds from a Pa~ticle Surface", which is Lncorporated hereiIl by reference. Suc~ removal S may be particula~ly advalltage~us prior to characte~tion, as discussed above.
Within a~other aspect of the invention, a method for er~iching fetal progenitor cells ~om mate~nal blood is provided comprising the steps of (a) incubadng a sanlple of mater~al blood wi~h axl immobilized ligand capable obinding ~o fetal progenitor cells under conditions, and for a time suf~cient to allow 10 specific binding of the ligand to the cells, and (b)removing u~bound blood products such that the fetal progenito~ cells are enriched. Within the context of the present iIlvention, fetal progenitor cells includes totipotent hematopoieticstem cells as well as early progenitor cells such as colony forming cells (CFCs).
Representative examples of CFCS include CFIJ-E, CFIJ-G, CFU-M, CFU-GM, 15 CFIJ-GEMM and BF~J-E cells. Given the ~act that generally as many as one-third to two-thirds of CD 34 positive cells may be stem cells or colony-~orming cells, it should be understood that when CD 34 ceL~ are ~oncentra~ed or purif;ed, fetal progenitor cells a~e lik~wise unders~ood to be concentrated or purified~
As noted aboYe, the sample of maternal blood is Lncubated with an 20 immobilized ligand c2lpable of binding to fetal progeDitor ce}ls. Wi~hin the context of the present invention, a ligand '~inds to" fetal progenitor cells if it :: recognizes a~ tigen associated with either both adul~ d ~etal cells or only fetal ;~ ~ progenitor cells. A representative antigen in ~lhis regard is the (:D-34 antigen.
Representative examples of antibodies which spe~;~cally recognize tbe CD 34 25 aIltigen include~ 10 aIld lHP~2, (BeCt~n-D1C1~nSOn~ MONntain View, Calif.~, QBEND-10 (Quantum Bi~systems, ~ambridge, U.K) and 12~8 ~Cellpro~, Bothell, W~h.). ~ ligaIld may be utilized in the above~described devices and methods in order to in~obilize tbe i~etal progeDitor cells. After incubation, the unbound blood products are removed as described above, ~uch that the ~etal progenitor 30 cells are enriched. Within the context of the present invention9 cells are : ~ "enn~hed" if gre~ter than 0.001% of the cells a~e fetal progeI~itor cells. Preferably, ~:~ the fetal progenitor cells are ennched to greater than .1% and particularly, to ~: greater than 1~ of the cells present.
Within another aspect of the preserlt invention, a method is 35 provided for en~ching fetal progen:itor celis from maternal blood comprising the steps of (a) incuba~ing a sample of mate~al blood with a labeled lig~nd capable of specifically biIlding to ~etal progerlitor cells under conditions and for a time .. . . .
.... . ..

.. .

W~3 93/~)X2~9 PCr/U!;92/a~024 ` 16 slaffi~ent to allow specific binding of the liga~d to the cells, (b) dete~g the prcse~cc of the ligant bou~d cells, and ~c) scpara~ tbe ligand bou~d cells from the unbo~d cells, such that thc ~tal proge~tor cells are e~r~ched. Wi~hi~ this aspect of the ~nYcntioD, ceL~s are ~Ilaubated ~nth a liga~ld sucb as that descrl~ed S abovc w~ich is c~able of spccifically bindi~g to fe~l progcnitor cc~ls, except that rathcr thaIl bcing ~obilized, this liga~d is labelcd. Yarious Iabcls may be u~lized withi~ the co~sext of the pres~t ~c~io~, although ~uorescem-isothocya~atc? phycoe~, rhodam~e isothios:yanate, or other such highly fl~uoresce~t molccules are par~cularly prefe~Ted.
1~ l~ough usc of ~ow cytometry (FACS) labeled ceLs may then be dete~ed, and separated from no~-labeled (or a n~n-ligand bou~d) cells, such that.etal progcnitor ceLIs are e~Tiched.
Within o~e embodimcnt of the i~re~ion, prior to thc step of incubati~g, red blood cells may be removed from m~ter~al blood, for example, by 15 ~g the materIlal blood over a Ficoll ~radie~ Withi~ another embodim~t, subse~ue~t to the step of rcmo~ be ~bou~d blood produc~ thc bound cells arc incubated i~ the prescnce of e~hropoie~n u~li~g culture me~hods des~bed ab~c.
Wi~n a~o~er aspec~ of tbe ~v~ on, a method is provaded for 20 e~nchi~g fetal p~ogeni~or ceL~ m mate~ blaod, compnsi~g the steps of (a) i~cuba~ng a samplc of mat~n: al blood wi~ i~obilized ligand capable of spec~ically bi~g to ceLs o~her than fetal proge~itor c~lls under condi~iorls andfor a ~me suf ;cieat to ~ow specific bi~ding of thc ligand to the othe~ cells, and (b) remo~i~g the nonbound ~tal progen~or cells, such ~hat the fe~ progeD~tor 25 cells are e~ichcd. As ~oted abo~e, CD 34 is a cell-surface an~gen that may befou~d o~ f~tal proge~itor cells. Ma~y ce~face a~gc~s howc~vcr, can not be found o~ proge~or ceLs, aIld thu~ maybc udlized to dcp}et~ a samplc of matcmal bl~d ~rom ccLl othcr than fetal proge~tor c~lls 3Rcpresc~ ve examplcs of such arl~igeDs ~cllldc Ia, Glycopho~ CD 3, CD 19, CD 11, CD 1~, CD 33, and 30 CD 45. Iig~ds, such as a~ibodies, whieh specifically bind tO such an~gens maybe purchased ~orrl commercial supplie~s such as Becto~-Dic3c~soD, Mountain Vi~w, Calif.
Within one cmbodime~t of this mven~o~ prior to the s~ep of incuba~g, red blood c lls arc removed ~um mater~al blood ~, for example, 35 n~i~g the maternal blaod over a Ficoll gradient.

1~:

W(~ 93/08269 2 1 2 2 1 ~ 8 PCr/US92/U9024 USBO~FErAL CE~LLS

As described herein, enriched fetal cells have a Yarie~ of uses. For example, through in si~u hybridization the presence of a selected genetic material 5 may be detected in a fetal cell. Similarly, DNA or RNA amplification m~y also be used to detect a selected genetic sequence in fetal cells. The methods of the present invention are partic~arly useful for fetal cells which are enriched to such an ~tent that they are ~menable to chromosomal typing by conveIltional cytogenetics techDiques wherein spreads of the cells' chromosomes a~e examined 10 under a rnicroscope.
In sih~ hyb~id~tion may be used as a method for detecting the presence of a selected genetic material within cells. See Pin:kel et a~, Proc Natl.
Acad. Sci U5A 85:913842 (1988); see a~so Hopman et al, "Detection of Numerical ~hromosome Aberrations in 13ladder Cancer by In Sitll Hybridization," Am. J. of 15 Pa~h 135(6):1105-1117 ~1989~. Briefly, the genetic material associated with the preferentially enriched cells is first exposed usi~g teehniques well known in the art. The genetic material is then incubated with a labeled probe capable of specifically hybridizing to ~he genetic material under eonditions alld for a time SUf~CieI1t tO a11OW hY~ atiOn tO OCCUr. See, for eXamP1e, K E. DaVieS, ~uman 20 Genetic JDise~ses, IRL PreSS, V~(3~, U.K (1986). Fina11Y, the PreSenCe Of thehYb~idLZed 1abe1ed PrObe iS deteCted. Within a Preferred embOdiment~ the genetiCmateria1 iS den~tUred after the SteP Of eXPOSing. Wit11in the COnteXt Of the PreSent inVen~on:~ genetiC materia1 inCludes WhOle Chr~mOSOmeS, DNA and :RN~
; ~ ~ V:ariOUS ~etbOdS are alSo We11 knOwn jn the art fOr amplifying and 25 deteCting genetiC materia1. FOr eXamPIe, if PreSen~, a Se1eCted genetiC SeqUenCe maY be amP~ ed USiDg te~hniqUeS We11 knOwn in the art, alld then PrObed fOr presence of that se~uence. See ~og~ et al, "1~ Improved Me~hod For Prenatal Dia~osis of Genetic Diseases by ~alysis of ~nplified DNA Seguences," The New Eng. J. of A~e~ 317~16):985-990 (1987); see also Witt and Erickson, i'A Rapid 30 Method for Detection o Y~Chromosomal DNA from Dried Blood Specimens by ~e Polymerase Chain Reaction," Human Genet. ~2:271-274 (1989). Methods for amplification include Polymerase Chain Reaction ("PCR") (see Mullis et al, U.S.
Patent No. 4,683,195; Mllllis et ~, IJ.S. Patent No. 4,683,202; and Mullis et a~, U.S. P~erlt No. 4,800,159, which are incoIporated herein by reference), and RNA-3~ based ampli~cation techniques. See Iizardi et al, Bio/'rechnology 6:1197-1202(1988); Kramer et a~, Nature 339:401-402 ~1989); and Lomeli e~ ~, Clinical Wo g3/~82~9 Pcr/uss2/o9o2~
212213~ ~
1~

Chemistry 35(9):182~1831 (l9g9); see also Kramer et al, U.S. Patent No. 4,786,600, which is inco~porated herein by refere~ce.
PCR is the most commoDly used method for ampli~g DNA
sequences. Briefly, amplification entails adding the appropriate primer(s), 5 er~mes aIld ~ucleotides i~to a reaction mix~ure, ~ollowed by several (2~80) s~ycles ~f denatura~ion and ~nealing i~ order to ampli~ the small amount of ta~get DN~ The DNA mixture is then separated by electrophoresis and hybridized with a labeled probe to detect the presence of the target sequence ofDN~
The preferentially enriched fetal ce}ls may also be chromosomally typed. See ~Iuman Cytogenetics, D.E. Rooney and B.H. Czepulkousl~ (edsO), IRL
Press, (:)xford, U.K (1986). Briefly, within a preferred embodiment, a sample containing at least 1 fetal cell in 105 other cells is cultured for 2-6 days in Iscoves' Modified Dulbecco's Medium (IMDM) (Gibco, Grand Island, N.Y.) contair ing a 15 final co~ceIItration of 20% ~etal bo~7ine serum (FBS) (HYCLONEn', Log~ IJtah)and 2 U/ml of highly pu~i~ed recombinant erythropoie~in (Terry Fox Laboratoly, Vancouver, B.C., ~da) i~ order to increase the proportion of cells in metaphase. The cells are then incubated in colcemid~ fixed, attac~2ed to mi~oscope sl;des, t~psin-treated and stained with Wright's stain. The slides may20 then be sca~ned microscopically for abnormal chromosomes.
~: Preferen~dally enriched fetal cells may also be utilized therapeutically. For example, within one aspec~ of the invention, a gene may be inserted into a retrovirus, and the re~ro~nrus utilized to infect fetal cells which are de~ciellt in this gene. The infected fetal cell may then be administered to the 25 fetus ~or another indi~idual) in order to remedy the genetic de~ect. Diseaseswhich may be treated in this manner are mlmerous, including for example ADA~
sickle cell ~mia, Thalassemia, a~d S~. Methods for inserdng genes into retro~riruses are also well documented (see, for 0~unple, WO 90/01870, WO
88/03167, WO 88/09670, WO 89/07150~ WO 89/11539 a~d WO 89/09271, all of 30 which are incorporated by refere~ce).
Fetal cells may also be utilized as universal donor cells. Briefly, because fetal progenitor cells do not c~eate as strong an immune reaction in recipients as do progenitor cells ~om adults, they can be transplanted with fewer side ef~ects. These fetal progenitor cells can thus be used for transplant to correct 35 inherited metabolic diseases in recipients. By expanding their rlumbers in culture, these fe~l progeI~itor cells may even be used in place of a bone marrow transplant following myeloblative therapy for ~ancer.

w~ 93/08269 2 1 2 ~ ~ 3 ~ P~r/US92/09024 Preferent;ally7 enri~hed fetal cells may also be made oncogenic ~n order ~o stud~r the pro~ess of such oncogel~ic cells, or ~n order to simplify e~ansion and maintenance of fetal cells for rese~rch or diagnostic purposes.
Represen~ative traIIsorming ge~es that may be ~erted i~to fetal cells (for S example, by retro~ses as disa~sed above) include SV40, Ti, myc, ras and src.
Preferentially, emiched ~etal cells may also be cryopreserved for future researeh, or i~or ~uture therapeutic use. BAefly, methods for freezi~g stem cells are descnbed in a pendi~g application en~tled "Method for ~eezing e~lgrafting c~lls" (At~orney~s Docket No. 200072.409). These methods may also be10 utilized to ~eeze fetal progenitor cells.
The following examples are offered by way of illustration~ and not by way of limitation.

EXAMPLES
E~LE 1 Carboxylation of a Polyac~ylamide Gel Seventee~ grams of dIy Biogel P-60n' ~5~100 mesh (wet), coa~se 20 beads) (BIORAD, C~atalog No. 15~163Q Richmond, Cali~ are added to 1.5 1 of 0.5 M N~ICO3/0.5 M Na2CO3. The pH is adjusted ~o 103 with NaOH and care~ully stirred with a mixer ~R:ZRl~ C~famo, Wiarton, Ontario, Canada) so asi not to damage the beads for appro~mately 20 to 30 ~utes. The mixture is then placed iIl a 6ûC water bath. After the mixture reaches a temperature of 60C, it is 25 incubated for an addi~onal 2 hours (at 60~C) with occiasional stirring. The mixture is then remoYed from ~he water bath, and placed iI~ an ice bath to bring the mixture temperature down to room temperature.
The beads are washed several times with distilled or deionized water, followed by severial wash~gs of PBS using a coarse glass filter connected to 30 a vacuum. ~he ~rboxylated gel may be stored iIl PBS at 4C~ and is stable for up ~o one year if sterilized or sgored with a preservative.

WO 93/~8269 Pcr/U~92/~9024 2122 1~8 ~ `~

~XAMPLli 2 Avidin Conjugating the Carba~ylated Biogel PBS is first removed ~om a measured amount of ~rboxylated S Biogel by filtering with a coarse glass filter connected to a vacuum. ~lle gel is then equilibrated iIl distilled or deionized water for 15-30 minutes. EquilibratioIl in wat~r causes a~ expa~sioll of the gel to a volume of about 4 times its previously measu~ed amount. The gel is resuspended in 10 ml of distilled or deio~ed water for each ml of gel ~as originally measured i~ PBS~.
T~ mg of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC-HCl) ~Sigma Chernical Co., Catalog No. E7750, St. Louis, Mo.) is added ~or each ~ o~ gel as originally measured. The pH is rapidly adjusted to 5.5 by dropwise addition of HCl. Care is taken to maintain the pH at 5.5; pHs of less tha~ 5.0 or greater than 6.0 result in significantly less a~vation of the Biogel. The : 15 mixture is stirred fo~ five mi~utes.
Avidin (International En:ymes, Inc., Fallbrook, Calif.) is diss~lved at: a conceIltration of be~veen 10 ~nd :100 mg~ml in deioDized water. Next, 1000g ~f avidin is rapidly added for each ml of gel (as origiIIally measured in PBS~.
The:~mixhlre is stirred for 1.5 h~rs. N~xt, 2 M glycine is added to give a final20 conce tration ~0.2 M glycine in the~ nure and stirred for an additional 1 hour.
~ be gel is washed with several volllmes of P~3S using a coarse glass er~ a~d vacuum, and stored in PBS at 4~. The gel is s~able for ~pproximately one year.
, ~
EXAMPLE ~
Immunoadsorption of Maternal Cells With The Two-Step Method , ~
P~PARATION OP ( ~ELLS
Twen~ milliliters of blood is obtained rom a pregnant female and 30 su~e~ded in ~ equal volume of PBS with l~o Bo~ine Serur~ umin (BSA) (Sigma Chemical CO.? St. Louis, Mo.~ in four 50 ml centrifuge tubes. ~he number :: : of nucleated cells/ml i~ counted in the A~ollowing manner A~ 50 ~1 aliquQ~ of mixed3 articoagulated whole blood is diluted into 3 mA of a 3% acetic acid SolutioAA. A~fter VOA texAllg~ 7 ,ul samples OA dAluted blood are loaded irto each of : 35 t~vo chambers of a: hema~ytometer. After about 3 minutes to aAlow settling of the cells, the nuclei of cells, which are almost all lysed by the acetic acid, a~e counted in the f~ur ruled A~Aelds per chamb~r of a hema~ometer with improved Neubauer WO 93/0826~ 2 1 2 .~ 1 3 8 Pcr/vss2/o9o24 rulings (VWR Scientific, Sa~ Frallcisco, Calif.), each field represendng 0.1 x 10-3 ~1 sample volume. The average number of nuclei per field is multiplied by a dilution factor of 61 x 104 to calculate the number of nucleated cells per rnilliliter of whole blood. If the average is less than 10 nuclei per ~eld, the procedure is5 repeated, except 50 ~l blood is diluted into only 1 ml acetic acid, with a resulting new dilution factor of 21 x 104.
Each tube of diluted blood is underlayered with 5 ml of Histopaque 1077 (Sigma Chernical Co.) and centrifuged at 700 x g for 15 minutes at room temperature. Cells at the interface are collected and washed once in PBS plus 1 10 BS~ l~e pellet is resuspended in 100 ~l in PBS plus 1% BSA.
Twenty micrograrns/milliliters of anti-trallsferrin receptor antibody (Becton Dickinson, Imrnunocytomet~ Systerns, Mountain View, Cali) is added to the rnixture and incubated for 15 minutes on ice. The cells are then washed once with 4 ml of PBS plus 1% BSA and centrifuged at approximately 400 x g for 515 minutes.
The cells are then gently resuspended to 1 ml and 1 ~g/ml of ~: biotinylated goat anti-mouse IgG (Southern Biotech, Bi~ngham, Ala) is added.
` The mixture is incubated for 15 Il~inutes on ice and wasbed twice as described~: ~ above with PBS plus l% BS~
: PRBPARATION OF COLUMNS
Carb~xylated Biogel P-30~ (prepared as descnbed above) is allowed to e~uilibrate to room temperature and placed in a K9/15 column (P.harmacia, Piscataway, NJ.) to a total~bed~ height of 1 cm. The column is washed with PBS, 25 followed by washes with PBS plus 5% BS~ This column functions as a "pre-colu~n." The avidin:column contains avidin-conjugated Biogel P-60~, which is prepared~ as described :above. The avidin-conjugated Biogel is allowed to equilibrate to room temperature and placed in a K9/15 column to a total bed height of 4 cm. The column îs then washed with several volumes of PBS, followed 30 by washes with PBS pIus 5% BSA.

IMMuNoADsoRprloN O~ CELLS
~ ~ Cells which have been prepared as descr~bed above are resuspended : ~ in PBS plus 5% BSA to a volume of 1 ml. The cells are then gently transferred ::
35 onto the top of the gel bed of the pre-column ~ïlter. The cells ~re allowed to filter through the pre-column and are washed with 1 ml PBS plus 5~ BSA. A
peristaltic pump (Cole-Parmer, r~ock~ord, Ill.~ controls flow from the avidin WO 93/~8269 PCr/US92/09~24 2 1 2 2 ~ ~3 8 r æ

column to a rate of abou~ 1 ~al/~ute. Once the cells have almost run down to the top of the avidin column bedS 1-2 ml of PBS p~us 5% BSA is added to the top of the avidin column in order to wash out remaining cells. The columD is washed out with 4-6 ml of PBS plus 5% BSA, followed by 4-6 ml of PBS.
S ..
lRBMOV~L OP ADSORB8D ~llS FR(~ THE~ A~DIN COLUMN
The avidin col~ is placed on top of a 15 ml centrifuge tube. The valve of the column is opened and 15 ml of RPMI 1 is added to the column with a wide bore, 9-inch transfer pipette. The RPMI is added to the column while the 10 pipette is used or mecbaI~cally agitating aIld resuspexldi~g the cell bed, thus allo~g cells to become detached from the gel matrix, and to filter into the eentrifuge tube. ~e tube is then centrifuged at 400 x g for 5 minutes and resuspended in cell culture media as described below.

XA~PLE~ 4 Prefere~tial Emichment of Fetal Nucleated Elythroid Cells ENRICHM~Nr OP FETAL CEL~; W1TH ER~H~OPOIEI1N
(:ells which are separated from the Avidirl-Biogel column above, are 20 resuspended iII Iscoves' Modiiïed Dulbecco's Medium (IMDM) (Gibco, Grand d, N.'Y.~ containing a fin~ centration of 20% fetal bovine serum (FBS) (~CLONEn', Logan, Utah), and 2 U/ml of highly purihed recombinant eryt~opoie~ (Tersy Fox Iabora~ory, Vancou~er~ B.C., Ganada~. Cells are diluted to S x 106 ~ucleated cells/ml and 200 ~l is plated into each well of a 96 2S well tissue culture plate w~th round bottorns ~Corning Glass Works" CorniIlg, N.Y.).

EN~CHM~Nr OF FE~rAL C~, BY AMMONIUM ION D~eRENrlATION
Cells which are separated from the Avidin column above are 30 ad3usted to a concentra~ion of less than 2 x 107 nucleated cells/~ . One volume of the cell rnLx~ure is chilled, arld a solution at 29C containing 18 volumes of 0.1844 M NH4Cl and 2 volurnes of 10 ~M acetazolamide is added. After 2 n~inutes, 2 volumes of 3mM NH4HCO3 is rapidly added, and the whole mixture gently stirred for 3 minutes. Cells are washed several times with PBS by centrifugation in 35 order to remove cellular debris.

WC~ 93/08269 2 1 2 2 1 3 8 Pcr/US92/09024 ~k~
In Si~u ~yb~idiza~on E~ched cells are exposed to a hypotonic solution ~.075 M KCl) for S 12 ~utes at 3rc. The tub~s are inverted once d~g the incubation to keep the cells suspended. Twen~ drop~ of freshly prepared fixative (3:1 meth~ol:acetic acid) is added to the cells, vortexed, and then centrifuged for 8 minutes at appro~mately 250 x g. Fresh fixative is added to the cells, followed by incubation for one hour at room temperature. The cells are centrifuged for 8 minutes at 10 approximately 250 x g. Fresh f;xative is added and the process is repeated one more time. Finally, the cells are resuspended in a small amount of fixative and placed at 4(: ~rernight.
The next day the cells are vortexed and placed onto microscope slides (Baxter, McGaw Park, 111.) which have been cleaned ~th etha~ol and 15 dipped i~ distilled water. ~e slides are allowed to dly for two days at room temperature.
The slides a~e beated in 70% ~ormamide in 2 x S~ (0O30 M NaCl, Q030 M Na c~rate) to 68C-70C ~or 2.5 min~tes. l'he slides are then imme~iatelyplaced in: a ri~se of 70% ethanol in water. Following the r~llse, th~ slides are20 plaoed successively in; 7û~, 95%, aIld 100% ethanol solutions for 5 n~inutes each.
Each alcohol solution mllst be maiIltained at ~ -2~(:~. The sl~des a~e air dried.
A probe is prepared from plas~d DNA according to the method of Page et al ("Single copy~ sequence hybridizes to polyn~rphic and homologous locion human X and~Y: ch~omosomes," PNAS 79:5352-5356 (1982), from American :: 25: Type ul~ e Collec~io~ ~(ATCC) No~ 57261; e~cept that biotin-dA~ is incolporated into the probe. Twent~ ve microliters of the bioti~yla~ed probe (5 ~egjml) per slide is placed in a microfuge ~ube. The probe is heated to 7QC for S
minutes the:n immedia~èly placed on ice Twenty microlite~ of the probe solution , ` is placed o~to e~ch slide and covered with a 22 x 40 mm coverslip. 'rhe slides a~e 30 placed into a box wî~h a wet paper tnweling liner and incubated at 37C for 12-18 hours.
A 50% solution of formamide in 1 x SCC is wanned to 37C.
C~verslips are removed fro~ the slides and immersed into the 50% ~ormamide solution for 30 minutes. The slides are then placed in 2 x SCC solution for 30 35 minutes with gentle rocking, then in 1 x SCC for 30 minutes with gentle rocking.
FluoresceiIlated avidin (Ve:ctor, Burlingame, Calif.) is diluted 1:1000 (1 l~g/ml~.
The back of the slide and around cell area are w~ped. Two hundred microliters of WO 93/08269 Pcr/u~92/09024 2122138 ~`

the aYidin-fluorescein is added to each slide and incubated in the box for 30 ~utes at room temperature. The slides are rinsed sequentia~ly in (1) 4 x SCC
~or 10 minutes with rocl~g, (2) 4 x SCC, 0.1~o Tween-20 for 10 m~mltes ~ithout rocking, and (33 4 x SSC for 10 ~utes with rocking. The ~ack of ~he slide aIld 5 ar~und cell a~ea is wiped o~. Ten microliters of anti-fade plus propidium iodine ~10 ml PBS, 100 mg p-Phenylene di~e, 90 ml glyceroL pH 8.0, 10 ~g/ml propidium iodide) is added. The slides are covered with coverslips and stored at4C The slides may be stored ~or several days if necessaly. Target DNA may be observed under a microscope by the presence of fluorescence.

Chromosomal Typing The enriched cells are exposed to 1 ~g of colcemid (Sigma, 15 St. Louis, Mo.) for one hour at 37C. A hypotonic solution (0.075 M KCl) is added to the cells and incubated ~or 12 minutes at 37C. The hlbes are inverted once during the incubation to keep the cells suspended. Twen~ drops of freshly prepared fixative (3:1 metha~ol:acetic acid) is added to the cells, v~r~exed, and the~ cent~ifuged for B minutes at approximately 250 x g. Fresh ~;xative is added to 20 the cells, followed by incubation for 1 hour at room temperature. The cells are ce~tri~uged for 8 ~utes at approximately 250 x g and fresh fixative is added again. This process is repeated one more time. Finally, the cells are resuspended in a small amou~t of fixative and placed at 4C o~ernight. The next day the cells e vorte3~ed and placed onto microscope slides ~Ba~ner, McGaw Park, nL) which 25 have bee~ cle~ ed with e~ha~ol ~ d dipped in distilled water. The slides are allowed to air dry ~or about 34 days, ia~d then are treated with 0.005% ~Iypsin (Diico ~actot~psirl, VWR Scientific, San Franasco, Calif.) ~r 30 to 35 secondsi.The slides iare washçd ~vice in PBS Plus 1~o FBS7 followed by wiasihiIlg in PBS
vnly. The cells are stained w~th Wright's solution (Sigma Chernical Co., St. Louis, 30 Mo.), followed by t-vo washes with deionized water. The slides are scaDned for evidence of metaphasie cells and ~ped by conventional cytogenetics.

wo g3/08269 2 1 2 2 1 3 8 P~r/uss2/o9o24 - Culb~re Without Sep~ation ~ ~aATQ~NALBLOOD
S Maternal samples were diluted 1:1 with Phospbate Buffered Saline (PBS). TweIl~y milliliters were placed in a celltriuge tu~e, foll~wed by 8 ml of Ficoll ~Iypaque. ~e tube wa~ ce~trifuge for 15 ~utes at 1700 rpm ~SOQxg).
The pelleted cells were washed twice by resuspeIlding and then centrifuging the.cells. ~e cells were resuspended in 20 ml of IMDM culture media eo~t~ng 10 20% Fetal Bovine Serum ~HYCLONE, Logan, Utah), 2mM glut~e, lmM
sodium pyruvate, 0.1 mM NEAA (non-essential amino acids; Whittaker Bioproducts, Walkersville, Md.)7 and 1 U/ml EPO ~Terry Fox Laboratory?
Vancouver, B.C., Ca~ada. Ten milliliters of the cell suspensiorl was placed intoeach of tWQ T'75 Costar 1asks, and incubated overnight at 37C in a 5% CO2 15 in~ubator. Adherent celIs such as ~lbroblasts are thus removed prior to separation.

EXA~
:: Separation of Fetal Progenitor Cells on an Affinity Column 20~
Samples from four patients were treated as described above in Example 7. ~er incubation ove~ight, the eell culture was counted and : ~ centri~uges 10: minutes ~t 1200 rpm. The supernatant was removed, and the cells wer~ resuspe~ded in 10 ~ of PBS containing 1~ BSA (Bov~ne Serum Albumin~
5 and centrifuged once more ~for 10 minutes at 1200 rpm (250 x g). The supernatant was removed and the cells~were resuspended in 1 ml of PBS containing 0.1~o BS~
Antibody 12.8 ~(CellPro, Bothell, Wash.) (an anti-CD 34 antibody~
was added to the cell suspension to a final concentration of 40 llg/ml. The cells were then iIlcubated for 30 minutes on ice.
30 ~ ~er 30 minutes, the cells were washed ~wice by ce~trifug~tion arldresusper~sio:ll in PBS/1% BS~ Next, biotinyl~ed }abbit anti-mouse IgM antisera (Zymed Laborat~ries, South San Francisco9 Cal.) was added to a final concentra$ion of 1~ 0 and~ incubated for 30 minutes on iceO The cells were washed twice by centrifugation aIld resuspension in PBSl1% BSA, and ~nally 35 res~spended in 1 ml of PB~ containing 5% BS~
An avidinated gel was prepared essentially as described il~ Examples 1 and 2 above, and placed into a scft column (see pendiIlg application U.S. Serial WO 93/~826~ 2 1 2 2 1 ~ 8 P~r/uss2/o9o24 No. 07/5gg,796, which is hereby inco~porated by reference) to a bed depth of 4 cm.

~2 S Culture Post Separa~don ~ dsorbed ~ells which were sepa~ated by the above procedure were resuspended in IMDM containi~g 20% FBS and 2 U/ml EPO. The cells were.
then placed in 9~ well Costar plates in a volume of 200 ~1 per well ~approximately 10 one million cells per well), and ~ncubated for 5 days at 37C in a 5% C02 incubator.

PR~PARATION OF CELLS FOR MErAPHAS~ SPR~ADS
After incubating for five days, the cells were transferred into 15 microcerltrifuge tubcs and exposed to Colcemid (1 ag in 300 ~1) for one hour at 37C. I'he cells were then:centrifuged for 5 minutes a~ tO00 rpm, and 1 ml of 0.~75M 1~(:1 was added. ~ Twenty drops of freshly prepared fixative (3:1 : Methanol:Acetic Acid) was added to ~he cell suspen~sion, and the cells were ceotlif iged again for ~ minutes at 10~0 Ipm. OIIe ~ ~f f;xative was added a~d 20 the ~c~lls were left at room temperature ~r 1 hour. T~e cells were then Centrifu~ed for S minutes at 1000 ~pm, and washed three times in 1 ml of fixative, ~d left in~ 500 ~l overnight at 4~C. The next d~y metaphase spreads were :prepa~ed.

B. M~HASE S~R~DS~ (KarOtYPIng) The spreads were allowed to d~y seve~al days at room tempera~ure.
Next, the slides were placed::~in Dif~o Ba~tot~psin~ for one minute (6 drops/45 ml : PBS). ~e slides were ~insed in PBS containillg 1% ~S, and then in PBS only.
The slides were placed in fresh Wright's stain for 35 seconds, rinsed ~wice, and3~ scaDned under the microscope.

~: : EXAMPLE 1Q
Determination of C~FCs One ml per 35 rnm plate of Iscove's Methylcellulose (Terry Fox Laboratories, Ya~couver, British Columbia, Canada) supplemgnted with ~ mM L-glutamine and 50 ~g/ml gentamic.n was wa~med to 37C. Cells were plated in WO 93~08~9 2 1 2 2 1 ~ ~ Pcr/vs92/oso24 triplicate at 3-~old dilutions to improve the accuracy of the assay. The highestnumber of cells plated was 10S/plate ex~ept for colun~-purified cells wbich wereplated at 3 x 103 and less. The cells were spread evenly over the surface of each plate and then incubated in a humidified ~ncubator at 3rC with 55'o CO2 in air for S 10 to 14 days. Color~ies were counted if they contained more than 50 cells andscored as GFU-GM, BFIJ-E, or other (~g., CFU-GEMM). The mlmber of various ~pes of colonies were summed to E~ive the total number ~f colony-formingcells (~C).
~e results are summarized briefly in Table 1 below:

CFC Number , lS Pregnant Female Starting cells 8796 Adsorbed cells 613 Unadsorbed cells 1234 Non-pregnant female : Starling cells 319 ~ , Adsorbed cells 90 Unadsorbed cells 79 Male ~; :: Starting cells 618 :Adsorbed cells l9S
Unadsorbed cells When~ CFCs are: compared, it is evident that a significantly higher number of cells were obtained from the pregnant female, as compared to tbe non-30 Pregnant female or the male. Although it is possible that the increase in numberis due to mobilization of progenitor cells from the pregnant female's bone ~: marrow, at least a portion of the increase in progenitor cells is due to fetal progenitor eells in the mother's circulation.

WO 93/08269 P~/U~92Jû9024 .~,, ~k~
In S~ ~Iybndiza~on Fetal cells which had bee~ enriched as des~ibed above in Example 5 8 were subjected to in si~u hybridization utili~ng a co~ercially available Idt(Chromosome ua situ kit S1370, Oncor, Gaithersburg, MD). CFCs n~Lmber was also de~e~ed as described above iIl Example 10. The results are briefly set forth below in Table 2.
TABL~2 SELECrED RESULTS OF F ETAI, CELL SEPARATION FROM ~T~3R~AL
BLOOD FO~WED BY CULTURE AND IN 3ITU HYlBR~ ON
YVITH Y-PROBE
' 15 P~TIEN l GESTATION OElL ~ (Mi0iol~) CFC (Per 105 Cells) IN ~ AMNIO
. . WEER;~S START ADS UNADS START ADS UNADS or CVS
A ~ ~9~05 10 20 6~00 3 Male Malo : B 16 ~011 31 10 1450 1 Male Male C 16 350.11 45 2 1610 0 O Female : :
: : ~ E~P~E 12 ACS A~alysis Fetal cells which were enriched as descr~bed aboYe in Example 8 : ~ were~ submitted to analysis by F~CS. Briefly, ~pproximately 125,0~ puri~ed cells were di~1ide~ into two tubes. One tube received an IgG control~ and the second received ~Bend-10 ~n a~ CD 34 antibody) at a final concentration of 20 ~g/ml.
T~e tubes were incubated for 30 mimltes on ice, then washed twice with 4 ml of 1% BSA in PBS.
Both tubes were then treated with a 1:50 dilution of [FTIC-conjugatedl goat anti-mouse IgG, incubated for 30 minutes on ice, and washed with 4 ml of 1% BSA in PBS. After the final wash the cells were suspended in 200~1 of PBS and propidium iodide (1 ~g/ml) and analyzed on a FACScan ~Becton Dickinson).

WO g3/08~69 2 12 2 13 X P~/US92/09024 ~9 EXAMPL~ 13 Plucldng and RecultuIing Colonies IIIdividual fetal cell colonies were sterilely plucked ~om the 5 methylcellulose culture of Exa~ple 10. The colonies were placed into E~ r~O
media, a~d then placed back ints methylcellulose culture to obsene dif~eren~iation. From ~he single cell, a coloIly of clonal fe~al cells develops.C~onal fetal cells are identical, and thus may be utilized for assays wherein the ef~ect of multiple samples are to be tested on cells (ie., to establish lQ whether a compound is carcinogenic). Some of the cells may function as a control, while other cells may be subjected to the compound. Simila~ly, multiple assays may be pe~folmed in order to determine the sensitivity of a ~ell to a certain drug.
In addi~ion, the colonies may be screened ~r a desired response, aII~ clones ~ubjected to more detailed analyses.

From the foregoing, it will be appreciated that, although specific embodiments of the invelltioa have been described herein for ~he purposes of illustration, va~i~us modiiïcations may be made wi~hout dev~ati~g from the spirit 20 all~ scope of the invention~ Accordingly, the invelltion is no~ limited except as by the appended claims.

,

Claims (19)

Claims

1. A method for enriching fetal progenitor cells from maternal blood, comprising:
(a) incubating a sample of maternal blood with an immobilized ligand capable of binding to fetal progenitor cells under conditions, and for a time sufficient to allow specific binding of said ligand to said cells; and (b) removing unbound blood products such that said fetal progenitor cells are enriched.

2. The method of claim 1 including, prior to the step of incubating, removing red blood cells from maternal blood.

3. The method of claim 2 wherein the step of removing comprises separating the maternal blood on a density gradient.

4. The method of claim 1 including, subsequent to the step of removing said unbound blood products, incubating the bound cells in the presence of erythropoietin.

5. A method for enriching fetal progenitor cells from maternal blood, comprising:
(a) incubating a sample of maternal blood with a labeled ligand capable of binding to fetal progenitor cells under conditions and for a time sufficient to allow specific binding of said ligand to said cells;
(b) detecting the presence of said ligand bound cells; and (c) separating said ligand bound cells from the unbound cells, such that said fetal progenitor cells are enriched.

6. The method of claim 5 including, prior to the step of incubating, removing red blood cells from maternal blood.

7. The method of claim 6 wherein the step of removing comprises separating the maternal blood on a density gradient.

8. The method of claim 5 including, subsequent to the step of removing said unbound blood products, incubating the bound cells in the presence of erythropoietin.

9. The method of claim 5 wherein said label is selected from the group consisting of fluorescein-isothiocyanate, phycoerythrin, biotin and rhodamine isothiocyanate.

10. The method of claims 1 to 5 wherein said ligand is an antibody.

11. The method of claim 10 wherein said antibody is 12.8.

12. A method for enriching fetal progenitor cells from maternal blood, comprising:
(a) incubating a sample of maternal blood with an immobilized ligand capable of binding to cells other than fetal progenitor cells under conditions and for a time sufficient to allow specific binding of said ligand to said other cells; and (b) removing the nonbound fetal progenitor cells, such that said fetal progenitor cells are enriched.

13. The method of claim 12 including, prior to the step of incubating, removing red blood cells from maternal blood.

14. The method of claim 13 wherein the step of removing comprises running the maternal blood over a Ficoll gradient.

15. The method of claim 12 wherein said ligand is an antibody.

16. A composition comprising maternal blood cells and fetal progenitor cells, said fetal progenitor cells present in an amount greater than 0.001%
of the total cells.

17. A composition comprising maternal blood cells and fetal progenitor cells. said fetal progenitor cells present in an amount greater than 0.1% of the total cells.

18. A composition comprising maternal blood cells and fetal progenitor cells, said fetal progenitor cells present in an amount greater than 1% of the total cells.

19. The method of claim 5 wherein said label is biotin, and wherein the step of separating comprises incubating biotin labeled cells with immobilized avidin, thereby allowing separation of said ligand bound cells.