EP0610359A1 - A method for enriching fetal progenitor cells from maternal blood - Google Patents

Abstract

The method described consists of (a) incubating a sample of maternal blood with an immobilized ligand capable of binding to fetal stem cells under conditions and for a time sufficient to allow the specific binding of the ligand to the cells, and (b) removing unfixed blood products, so that fetal stem cells are enriched.

Description

Description

A METHOD FOR ENRICHING FETAL PROGENITOR CELLS FROM MATERNAL BLOOD

Cross Reference to Related Application

This application is a continuation-in-part of pending U.S. Application Serial No. 07/513,057, filed April 23, 1990.

Technical Field

The invention relates to a method of enriching fetal progenitor cells from maternal blood.

Background of the Invention

Approximately 5.3 million women become pregnant in the United States yearly, resulting in 3.8 million deliveries. There are an additional 10 million deliveries in the other affluent countries in the world. Prenatal testing is used in a subgroup of these women who have a significant risk of having a fetus with a genetic disorder such as Down's Syndrome.

At present, the only way of diagnosing fetal disorders is to obtain cells from the amniotic fluid (amniocentesis) or the surface of the fetal sac (chorionicvillus) of the mother. These procedures are expensive and carry a risk of spontaneous abortion of between V2 0 to 1%. Because of the risk of spontaneous abortion, these collection procedures are recommended only for women who are at a high-risk of carrying a child with a genetic defect. For example, only women over age 35 are advised to have the test because the risk of Down's Syndrome is higher in that group. Some of these women refuse the test because of the risk of spontaneous abortion. Even though many of these high-risk women are tested, only a fraction of fetuses with Down's Syndrome are detected. The high-risk women represent such a small portion of the women having children that the low-risk population still delivers most of the afflicted children. Eighty percent of those children born with Down's Syndrome are from the "low-risk," under 35-year-old group. This situation is also true of many other genetic defects or disorders. It is therefore desirable to provide a test that would resolve this testing dilemma by providing a safe method which could be available to all pregnant women, irrespective of the risk factor and without risk of spontaneous abortion. Although it is known that fetal cells circulate in the blood stream of pregnant women (see Kulozik and Pawlowitzki, "Fetal Cells in the Maternal Circulation: Detection by Direct AFT- mnunofhiorescence," Human Genetics 62:221-224 (1982)), fetal cells are present in such low concentrations that the procedures necessary to isolate them are extremely difficult and time-consuming. For example, Herzenberg et al, "Fetal Cells in the Blood of Pregnant Women: Detection and Enrichment by Fluorescence-Activated Cell Sorting," Proc. Natl Acad. Set, USA 75:1453-1455 (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 Hertzenberg et al is also deficient for routine clinical testing because it requires determination of HLA types. Bianchi et al "Direct Hybridization to DNA From Small Numbers of Flow-Sorted Nucleated Newborn Cells," Cytometry 5:197-202 (1987) also used a FACS machine to detect nucleated cells, although the blood sample was not obtained from the mother but from the newborn's umbilical cord. The present invention provides a method for enriching fetal cells that overcomes these disadvantages, and further provides other related advantages.

Summary of the Invention The present invention is also directed toward methods for enriching fetal nucleated, erythroid cells from maternal blood. Within one aspect of the present invention, such a method comprises the steps of (a) incubating a sample of maternal blood with an immobilized ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of the ligand to the cells, (b) removing unbound blood products, and (c) incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched. Within one embodiment of this aspect of the invention, the ύnmobilized ligand is an immobilized antibody.

Within another aspect of the invention, a method is provided for enriching fetal nucleated, erythroid cells from maternal blood, comprising the steps of: incubating a sample of maternal blood with a first member chemically linked to a ligand capable of specifically binding to fetal nucleated, erythroid cells 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, the second member being capable of binding to the first member with an affinity constant of greater than about 10° M^"1; removing unbound blood products; and incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched. Suitable first member-second member binding pairs include biotin-avidin, biotin-streptavidin, biocytin-avidin, biocytin-streptavidin, mefhotrexate-dihydrofolate reductase, 5-fluorouracfl-thimydylate synthetase, and riboflavin-riboflavin binding protein. Within one embodiment of this aspect of the invention, the first member which is chemically linked to a ligand is a biotinylated antibody and the immobilized second member is immobilized avidin.

Within another aspect of the present invention, the method comprises the steps of: incubating a sample of maternal blood with a first ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient 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 binding to the first hgand under conditions and for a time sufficient to allow the second Hgand to bind to the first ligand; adsorbing the cells to an immobilized second member, the second member capable of binding to the first member with an affinity constant of greater than about 10° M , removing unbound blood products; and incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched. Within one embodiment, the first hgand is an antibody which specifically binds to fetal nucleated, erythroid cells. Within preferred embodiments, the first member which is chemically linked to the second gand is a biotinylated antibody. In such an embodiment, the ύmnobilized second member is immobilized avidin.

As an alternative to the step of incubating bound cells in the presence of erythropoietin, another aspect of the present invention comprises the steps of: (a) incubating the bound cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within the bound cells, and (b) incubating the bound cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs. In addition, the present invention may also be performed using a combination of both erythropoietin enrichment and the method described above wherein the cells are incubated with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within the bound cells, followed by the incubation of bound cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs. These two enrichment methods may be performed sequentially, and in either order. Within another aspect of the present invention, a method is provided for enriching fetal nucleated, erythroid cells from maternal blood, comprising the steps of: incubating a sample of maternal blood in the presence of erythropoietin such that the fetal cells are enriched; incubating the enriched cells with an immobilized ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of the hgand to the cells; and removing unbound blood products. Alternatively, within another embodiment of the present invention, the enriched cells may be immobilized by incubating them with a first member chemically linked to a hgand capable of specifically binding to the cells under conditions and for a time sufficient to allow specific binding of the hgand to the cells, and adsorbing the cells to an immobilized second member, the second member being capable of binding to the first member with an affinity constant of greater than about 10° M^"1 . Within yet a further embodiment of the present invention, the enriched cells may be immobilized by incubating the enriched cells with a first hgand capable of specifically binding to the cells under conditions and for a time sufficient to allow specific binding of the first hgand to the cells; incubating the sample with a first member chemically linked to a second hgand capable of specifically binding to the first hgand under conditions and for a time sufficient to allow the second hgand to bind to the first hgand; and adsorbing the cells to an immobilized second member, the second member being capable of binding to the first member with an affinity constant of greater than about 10° M . Suitable combinations of first and second hgand are discussed in detail below.

In addition, within the aspects discussed above, the methods may further comprise (subsequent to removing the unbound blood products) the steps of: incubating the bound cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within the bound cells; and incubating the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide, such that selective hemolysis of maternal blood cells occurs. Within other aspects of the present invention, as an alternative to first mcubating a sample of maternal blood in the presence of erythropoietin, the cells are incubated with ammonia .and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within the bound cells, followed by incubation of the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide, such that selective hemolysis of maternal blood cells occurs. Within the present invention, a variety of ligands may be utilized, including antibodies, eiythropoietin, and transferrin, The ligand may be immobilized on any of a variety of sohd supports, such as hollow fibers, beads, magnetic beads, plates, dishes, flasks, meshes, screens, sohd fibers, membranes, and dipsticks. Within another aspect of the invention a method for enriching fetal progenitor cells from maternal blood is provided comprising the steps of (a) incubating a sample of maternal blood with an immobilized hgand capable of specifically binding to fetal progenitor cells under conditions, and for a time sufficient to allow specific binding of the hgand to the cells, and (b) removing unbound blood products such that the fetal progenitor cells are enriched. Within one embodiment, prior to the step of incubating, red blood cells are removed from maternal blood, for example by running the maternal blood over a Ficoll gradient. Within another embodiment, subsequent to the step of removing the unbound blood products, the bound cells are incubated in the presence of erythropoietin. Within another aspect of the present invention, a method is provided for enriching fetal progenitor cells from maternal blood comprising the steps of (a) incubating a sample of maternal blood with a labeled hgand capable of specifically binding to fetal progenitor cells, under conditions, and for a time sufficient to allow specific binding of the hgand to the cells, (b) detecting the presence of the hgand bound cells, and (c) separating the hgand bound cells from the unbound cells, such that said fetal progenitor cells are enriched. Within one embodiment, prior to the step of incubating, red blood cells are removed from maternal blood by, for example, running the maternal blood over a Ficoll gradient. Within another embodiment, subsequent to the step of removing the unbound blood products, the bound cells are incubated in the presence of erythropoietin. Within the embodiments, the label is selected from the group consisting of fluoroscein-isothiocyanate, phycoerythrin, rhodamine isothiocyanate, or other such highly fluorescent molecules. In other embodiments of the invention, the hgand is an antibody such as 12.8. Within another aspect of the invention, a method is provided for enriching fetal progenitor cells from maternal blood comprising, the steps of (a) incubating a sample of maternal blood with an immobilized hgand capable of specifically binding to cells other than fetal progenitor cells, under conditions and for a time sufficient to allow specific binding of the hgand to said other cells, and (b) removing the nonbound fetal progenitor cells, such that said fetal progenitor cells are enriched. Within one embodiment, prior to the step of incubating, red blood cells are removed from maternal blood by, for example, running the maternal blood over a Ficoll gradient. Within a preferred embodiment, the hgand is an antibody.

Within another aspect of the present invention a method is provided for typing chromosomes of fetal nucleated erythroid cells, comprising the steps of (a) incubating the fetal nucleated erythroid cells in a media containing erythropoietin under conditions and for a time sufficient to induce metaphase in the cells, (b) fixing the DNA of the cells, (c) staining the fixed DNA such that chromosomes may be observed, and (d) examining the stained DNA thereby allowing the typing of the chromosomes. These and other aspects of the present invention will become evident upon reference to the following detailed description.

Detailed Description of the Invention

As noted above, within one aspect of the present invention methods are provided for the enrichment of fetal nucleated, erythroid cells from maternal blood. Maternal blood contains, among many other types of cells, both adult and fetal nucleated, erythroid cells. Through the efforts of the present invention, fetal nucleated, erythroid cells may be enriched from as few as 1 in 10" in maternal blood, to an enriched concentration of about 1 in lC , and preferably, to about 1 in 10². Within the context of the present invention, nucleated erythroid cells contain a nucleus and generally include erythroblasts as well as other erythroid precursor cells.

Maternal blood may be obtained from a pregnant female using conventional techniques well known in the art. Preferably, peripheral blood is drawn from an easily 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 4°C for a maximum of 4 to 7 days. Various anticoagulants may be added to the blood as necessary, including, among others, ACD, CPDA, EDTA, and Heparin. The maternal blood is then subjected to a selection method in accordance with the present invention, wherein preferentially enriched fetal cells may be obtained without the need for further purification, for example, by a Fluorescence-Activated Cell Sorter (FACS). In general, the methods of the present invention comprise the steps of: (1) incubating the maternal blood with either an immobilized hgand, or a hgand which will subsequently be immobilized, such that the hgand binds to and hence immobilizes the fetal nucleated, erythroid cells, (2) removing unbound blood products, and (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, erythroid cells,

(2) incubating the enriched cells with either an immobilized hgand, or a hgand which will subsequently be immobilized, such that the hgand binds to and hence immobilizes the fetal nucleated, erythroid cells, and (3) removing unbound blood products. The present invention may be performed utilizing devices as described in an application entitled "Immunoselection Device and Method," U.S. Serial No. 07/513,543, and an application entitled "An Apparatus and Method for Separating Particles Using a Pliable Vessel," U.S. Serial No. 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. Apphcation (Attorney's Docket No. 200072.407) Serial No. entitled "Improved Apparatus and Method for Cell Separation" (hereby incorporated by reference in its entirety). Briefly, within one aspect of this apphcation, a "cell separator" is provided, including a column assembly for separating target cells from a sample fluid, the column assembly including a column, a sample fluid supply bag and a fluid coUection bag wherein the column is provided for receiving the sample fluid from the sample fluid supply bag and for separating the target cells from the sample fluid and retaining the target cells, and wherein the fluid collection bag is provided for receiving the target cells after being released from the column, said cell separator comprising an agitation means for agitating the contents of the column to assist in releasing the sample cells retained in the column, the agitation means being responsive to a drive signal for varying amount of agitation of the contents of the column to vary the rate at which the sample cells are released, column sensor means for providing a column signal indicative of the optical density of fluid flowing out of the column and into the fluid coUection 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 fluid coUection bag, and a data processor means for controlhng the operation of the ceU separator, the data processor means being response to the column signal for providing the drive signal and the column valve control signal to prevent inadequate concentrations of the target cells from being coUected. One embodiment of this invention is the CEPRATE LC™ ceU separation system which is avaUable from CeUPro^® (BotheU, Wash.).

Within one aspect of the present invention, the maternal blood is incubated with an immobilized hgand capable of specificaUy binding to fetal nucleated, erythroid cells under conditions, and for a time sufficient to aUow binding of the cells to the hgand. GeneraUy, incubation of about 15 to 30 minutes at 4°C to 37°C is preferred. If the incubation step occurs as the cells are passed, over a column, the flow rate should be sufficiently slow to aUow the cells to bind. Preferably, the ceUs should be aUowed at least 15 minutes in which to traverse the bed of the column.

As noted above, the hgand should be chosen such that it is capable of specificaUy binding fetal nucleated, erythroid ceUs. Within the context of the present invention, the hgand is defined to be "specificaUy binding" if it is capable of binding to fetal nucleated, erythroid cells, but not to more than about 10% of the maternal blood cells. The relative percentage of bound fetal nucleated, erythroid ceUs to other ceUs may be reach y determined by analysis with a specific marker such as Alpha Fetal Protein (AFP). For example, at any point within the methods discussed below, the relative percentage of fetal cells to maternal ceUs may be determined with either glucose oxidase or fluorescein conjugated anti- AFP antibody. See A Kulozik and I. H. Pawlowitzki, "Fetal CeUs in the Maternal Circulation: Detection by Direct AFP-Immunofluorescence," Human Genet. 62:221 (1982). This determination is preferably performed after two or more purification steps. Ligands which specificaUy bind to fetal nucleated, erythroid cells are known in the art, including erythropoietin (Amgen, Thousand Oaks, Calif.), transferrin (Sigma Chemical Co., St. Louis, Mo.) and selected antibodies. Monoclonal antibodies which specificaUy recognize nucleated erythroid cells are particularly preferred. Monoclonal antibodies to nucleated erythroid ceUs such as anti-transferrin receptor antibodies may be obtained from conventional supphers (Becton Dickinson Immunocytometry Systems, Mountain View, Calif.). Alternatively, monoclonal anti-erythroid antibodies, such as EP-1, may be produced using techniques weU known in the art. See Yokochi etal, "Monoclonal Antibodies Detecting Antigenic Determinants With Restricted Expression On Erythroid CeUs: From the Erythroid Committed Progenitor Level to the Mature Erythroblast," Blood 63:1376 (1984); see also Heddy Zola (ed.), Monoclonal Antibodies: A Manual of Techniques, CRC Press, Boca Raton, Fla. (1987). Briefly, ceUs may be generated for immunization from fetal liver clonal erythroid cultures, and enriched for progenitor ceUs. Thus, the population of cells used for antigens and for primary screening may contain immature erythroblasts, erythroblasts of an intermediate degree of maturity, and presumably, progenitor ceUs of BFU-E and CFU-E types. These cells may be used for intravenous immunization, foUowed by removal of the spleen and fusion of the spleen cells with a myeloma line such as NSI, using standard techniques. The resulting fused ceUs, or hybridomas, may then be screened against the above-described ceUs using conventional techniques. See Yokochi et al, supra. The entire specificaUy binding antibody need not be used as the hgand. More specificaUy, only the binding region of the antibody is necessary to specificaUy bind fetal nucleated, erythroid ceUs. Thus, antibody fragments such as Fab or F(ab')2 fragments may be used within the present invention. Additionally, the binding regions of the specificaUy binding antibody may be incorporated into a new protein, which may be used as the hgand. See Reichmann et al, "Reshaping Human Antibodies For Therapy," Nature 332:323-327 (1988); Verhoeyen et al, "Reshaping Human Antibodies: Grafting an Antilysozyme Activity," Science 239:1534-1536 (1989); and Roberts et al, "Generation of an Antibody with Enhanced Affinity and Specificity for its Antigen by Protein Engineering," Nature 325:731-734 (1987).

Within the present invention, the hgand is immobilized in order to aUow separation of bound cells from other blood products. Many suitable supports are weU known in the art and include, among others, hoUow fibers (Amicon Corporation, Danvers, Mass.), beads (Polysciences, Warrington, Penn.), magnetic beads (Robbin Scientific, Mountain View, Cahf.), plates, dishes and flasks (Corning Glass Works, Corning, N.Y.), meshes (Becton Dickinson, Mountain View, Calif.), screens and solid fibers (see Edelman et al, U.S. Patent No. 3,843,324; see also Kuroda et at, U.S. Patent No. 4,416,777), membranes (Millipore Corp., Bedford, Mass.), and dipsticks. A variety of different sources exist for supports other than those designated. Particularly preferred is a support such as Biogel P-60™ (BIORAD, Richmond, Calif.). Biogel P-60™ is a porous polyacrylamide hydrogel bead. The beads are generally spherical, on average about 250 microns in size, and have an average pore size which excludes molecules larger than approximately 60,000 daltons. A variety of methods may be used to immobUize the hgand onto a support. For example, a hgand, such as an antibody, may be directly coupled to the support by various methods wiU known in the art. See J. K. Inman, Methods In Enzymology, Vol.34, Affinity Techniques, Enzyme Purification: PartB, W. B. Jakoby and M. WUchek (eds.), Academic Press, New York, p. 30 (1974); see also M. WUchek, and W. Bayer, "The Avidin-Biotin Complex in Bioanalytical Applications," Anafyt. Biochem. 171:1-32 (1988). These methods include the use of glutaraldehyde, carbodhmide, carbonyl diimidazole, cyanogen bromide, tosyl chloride, biotin/avidin, and biotin/streptavidin. Once the hgand has been immobilized onto the support, the maternal blood may be incubated with the immobilized hgand under conditions, and for a time sufficient to aUow binding of the hgand to the ceUs. Within the context of the present invention, suitable conditions for binding to occur include incubation in a physiological buffer at about 4°C to about 37°C. Particularly preferred temperatures range from about 4°C to room temperature. The time of incubation depends on the affinity and avidity of the hgand for the ceU, and may be readUy determined. GeneraUy, incubation for about 15 minutes to one hour is preferred. FoUowing incubation, unbound blood products may be removed, and fetal ceUs enriched using the methods described herein.

Within another aspect of the present invention, a sample of maternal blood is incubated under suitable conditions with a hgand which is chemicaUy linked to a first member, and then adsorbed to a second member which is immobilized on a sohd support. The first member shduld be capable of binding to the second member with an affinity of greater than about 10° M . Many suitable first member-second member binding pairs are weU known in the art. These include, among others, biotin-avidin, biotin-streptavidin, biocytin- avidin, biocytm-streptavidin, methotrexate-dihydrofolate reductase, 5-fluorouracU-thimydylate synthetase, riboflavin-riboflavin binding protein, antibody-protein A, and antibody-protein G. In a preferred embodiment, the first member is biotin and the second member is avidin.

Either member of the above described binding pairs may function as the second member, with the complementary member functioning as the first member. Furthermore, combinations of the first member-second member binding pair may be employed. For example, biotin may be linked to the hgand, as weU as adsorbed to the support. The ceU, hgand, biotin-complex and biotin, support- complex may then be bound together through an incubation step with avidin. Avidin is multivalent, permitting the formation of a ceU, hgand, biotin, avidin, biotin, support-complex which immobilizes the ceU.

Within one example of this embodiment, a sample of maternal blood is incubated with a biotiny ted antibody under conditions and for a time sufficient to aUow binding to occur. The sample is then incubated with, or passed over, a support which contains immobilized avidin. CeUs which are coupled to the biotinylated antibody are adsorbed to the immobilized avidin, thus aUowing separation of ceUs from unbound blood products. Subsequently, unbound blood products may be removed, and fetal ceUs enriched using methods described below. Within yet another aspect of the present invention, a two-step method is used to immobilize the fetal nucleated, erythroid cells. Briefly, a first hgand is incubated with a sample of the maternal blood under suitable conditions as described above. Subsequently, a second hgand which has been chemically linked to a first member is added. The second hgand is capable of binding to the first hgand. The cell, first hgand, second ligand, first member-complex may then be adsorbed onto an immobilized second member, thus allowing the separation of cells from unbound blood products. Representative examples of the first member- second member binding pair have been discussed above. Representative examples of the first hgand include erythropoietin, transferrin and selected antibodies. Once the first hgand has been selected, the second ligand is chosen such that it specifically recognizes and binds to the first ligand. Within a preferred embodiment, the second hgand is an antibody, for example, an anti-erythropoietin (Terry Fox Laboratory, Vancouver, B.C., Canada), anti-transferrin (Chemicon Intl., Inc., Temecula, Cahf.), or anti-immunoglobulin antibody. Anti- immunoglobulin antibodies may be prepared using techniques weU known in the art, or may be obtained from conventional sources, including, among others, Sigma Chemical Co., St. Louis, Mo., and Becton Dickinson Immunocytometry Systems, Mountain View, Calif. Within a preferred embodiment, the first hgand is an antibody which specifically recognizes fetal nucleated, erythroid cells such as an anti- transferrin receptor antibody (Becton Dickinson Immunocytometry Systems, Mountain View, Cahf.). The antibody is incubated with a sample of maternal blood. A biotinylated anti-immunoglobulin antibody, such as biotinylated goat anti-mouse IgG (the second hgand which is chemically hnked to a first member) is then added and incubated with the sample. The sample is then incubated with, or passed over a bed of material which contains the immobilized second member, in this case, immobilized avidin. The ceU, antibody, anti-immunoglobulin antibody, and biotin-complex will be adsorbed to the immobilized avidin, thus allowing the subsequent removal of unbound blood products.

As noted above, once the ceUs have been immobilized, unbound blood products may be removed. In one embodiment, the immobilized ceUs are rinsed with a physiological buffer, thereby removing the unbound blood products. Various methods maybe used to rinse the immobilized ceUs, depending upon the type of support chosen. These methods include, among others: washing or flushing the support; magnetically attracting the support out of solution, foUowed by resuspension in a physiological buffer; and centrifugation foUowed by resuspension. Various physiological buffers are also weU known in the art, including PBS, PBS plus albumin, such as Bovine Serum Albumin (BSA), normal saline and ceU culture media.

Once unbound blood products have been removed, bound ceUs may be preferentiaUy enriched for fetal nucleated, erythroid cells. As noted above, at least two alternative methods may be used either separately, or together. If the two methods are performed together, either method may be performed first. Within one embodiment the bound ceUs are cultured under selected culture conditions in the presence of erythropoietin (Amgen, Thousand Oaks, Calif.). See Emerson et al, "Developmental Regulation of Erythropoiesis by Hematopoietic Growth Factors: Analysis on Populations of BFU-E From Bone Marrow, Peripheral Blood and Fetal Liver," Blood 74(l):49-55 (1989); see also Linch et al, "Studies of Circulating Hemopoietic Progenitor CeUs in Human Fetal Blood," Blood 59(5):976-979 (1982). Selected culture conditions generaUy include growth in standard ceU culture media, without any other cytokines other than erythropoietin. This preferentiaUy aUows fetal, but not maternal, nucleated erythroid ceUs to grow. Particularly preferred media includes Iscoves' Modified Dulbecco's Medium (Gibco, Grand Island, N.Y.) containing a final concentration of 20% fetal bovine serum, and 2 U/ml purified urinary human EPO. Within the second method, fetal nucleated, erythroid ceUs are preferentiaUy enriched based upon their uptake of ammonium ions and the selective hemolysis of maternal blood cells, or more specificaUy, of maternal erythroid cells. See generally Jacobs and Stewart, "The Role of Carbonic Anhydrase in Certain Ionic Exchanges Involving the Erythrocyte," /. Gen. Physiol 25:539-552 (1942); and Maren and WUey, "Kinetics of Carbonic Anhydrase in Whole Red CeUs as Measured by Transfer of Carbon Dioxide and Ammonia," Molecular Pharmacology 6:430-440 (1970). Briefly, the cells are incubated in the presence of ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to aUow accumulation of ammonium ions within the ceUs. This generaUy takes approximately 5 to 30 minutes. The cells are then incubated in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs. Within the context of the present invention, many compounds may provide suitable sources of ammonia, chloride ions and carbon dioxide. For example, suitable sources of ammonia include, among others, ammonia and the ammonium salts. Suitable sources of chloride ions include, among others, NaCl, KC1, MgCi2 and CaC - Suitable sources for carbon dioxide include, among others, carbon dioxide in solution, carbonate and bicarbonate.

In addition, many carbonic anhydrase inhibitors are known in the art, including, among others, most monovalent anions such as cyanide and cyanate, monovalent sulfides, sulfonamides, and acetazolamide. See Lindskog et al, The Enzymes 5:587 (1971); Ward and Cull, Arch. Biochem. Biophys. 750:436 (1972); and Pocker and Watamori, Biochem. 12:2475 (1973). The carbonic anhydrase inhibitors should be selected so as to function under physiological conditions.

Preferred carbonic anhydrase inhibitors include sulfanUamide and acetazolamide.

Bound ceUs may be released by various methods either subsequent to, or prior to enrichment. If bound cells are released prior to enrichment, they may be subsequently enriched using the above-described methods. Various methods are known in the art for releasing ceUs. Within one such method, ceUs may be cultured with or without cytokines. Cytokines, such as EL-2, can cause proliferation of cells, or changes in the surface characteristics of the cells, such that the ceUs or their progeny are released from a support. Within another method, cleavage of the hgand or of the ceU-hgand bond may release the cell. Various cleavable ligands and cleaving enzymes are known in the art, including among others, papain and trypsin. Within yet another method, the ceUs may be released by mechanical, gravitational or electromagnetic forces. A particularly preferred method is mechanical agitation, for example, by agitation of the beads through pipetting, stirring, shaking, vibration, or sonication.

Within another aspect of the present invention, a method is provided wherein the ceUs are first enriched, foUowed by adsorption of fetal nucleated, erythroid ceUs and removal of unbound blood products. Briefly, fetal ceUs may be enriched by first incubating maternal blood with ammonia and chloride ions, and a carbonic anhydrase inhibitor. After the ceUs have been incubated under conditions and for a time sufficient to allow accumulation of ammonium ions, the ceUs are treated with ammonia and carbon dioxide such that selective hemolysis of maternal blood ceUs occurs. Fetal cells may also be enriched by incubation with erythropoietin as discussed above, or by a combined treatment using both erythropoietin and the method discussed above wherein the ceUs are treated with ammonia and carbon dioxide. Furthermore, as discussed above, the two methods may be performed in either order.

The enriched cells remaining after the method(s) described above may then be immobilized using any of the above discussed methods, including: (1) an immobilized hgand which specificaUy binds to fetal nucleated, erythroid cells remaining after enrichment, (2) incubating ceUs remaining after enrichment with a first member linked to a Hgand, the Hgand being capable of specificaUy binding to fetal nucleated, erythroid cells, foUowed by adsorption of the cells to an. immobilized second member, the second member being capable of binding to the first member with an affinity constant of greater than about 10° M , and (3) incubating ceUs remaining after enrichment with a first Hgand, foUowed by incubation with a second Hgand which is chemicaUy Hnked to a first member which is capable of binding to the first Hgand under conditions and for a time sufficient to aUow the second Hgand to bind to the first Hgand, foUowed by adsorption of the cells to an immobilized second member, the second member being capable of binding to the first member with an affinity constant of greater than about 10⁸ M^_1 .

Subsequent to immobihzation of the cells, unbound blood products may be removed using methods described above. If the ceUs were initiaUy enriched using erythropoietin, subsequent to removing unbound blood products, the cells may be treated using the method wherein ammonia and carbon dioxide are used. Siπiilarly, if these cells were initially treated with the method wherein ammonia and carbon dioxide are used, subsequent to removing unbound blood products, the cells may be treated with erythropoietin. Fetal ceUs which have been enriched from maternal blood may be characterized by incubating the preferentiaUy enriched ceUs with a marker capable of selectively binding to fetal ceUs. A marker selectively binds to fetal cells when its presence on the target cells is greater than 10-fold more than the quantity on maternal cells. Various markers are known in the art, including, for example, antibodies to Alpha Fetal Protein ("AFP"). See Kulozik et al, "Fetal CeUs in the Maternal Circulation: Detection by Direct AFP- Immunofluorescence," Human Genet. 62:221-224 (1982), or antibodies to antigen "i". See Y.W. Kan et al, "Concentration of Fetal Red Blood CeUs From a Mixture of Maternal and Fetal Blood by Anti-i Serum," Blood 43(3):411-415 (1974). Antibodies to the fetal ceU marker may be labeled using techniques weU known in the art and used to detect the presence of fetal ceUs. Within a preferred embodiment of the present invention, Hgand is removed from the ceU surface through the method described in a related apphcation (U.S. Serial No. 07/513,056) entitled "Methods for Removing Ligands from a Particle Surface", which is incorporated herein by reference. Such removal may be particularly advantageous prior to characterization, as discussed above.

Within another aspect of the invention, a method for enriching fetal progenitor cells from maternal blood is provided comprising the steps of (a) incubating a sample of maternal blood with an immobilized hgand capable of binding to fetal progenitor ceUs under conditions, and for a time sufficient to aUow specific binding of the hgand to the ceUs, and (b) removing unbound blood products such that the fetal progenitor cells are enriched. Within the context of the present invention, fetal progenitor cells includes totipotent hematopoietic stem ceUs as weU as early progemtor cells such as colony forming cells (CFCs). Representative examples of CFCS include CFU-E, CFU-G, CFU-M, CFU-GM, CFU-GEMM and BFU-E ceUs. Given the fact that generally as many as one- third to two-thirds of CD 34 positive ceUs may be stem ceUs or colony-forming ceUs, it should be understood that when CD 34 cells are concentrated or purified, fetal progenitor ceUs are likewise understood to be concentrated or purified.

As noted above, the sample of maternal blood is incubated with an immobilized hgand capable of binding to fetal progenitor ceUs. Within the context of the present invention, a Hgand "binds to" fetal progenitor cells if it recognizes an antigen associated with either both adult and fetal cells or only fetal progenitor ceUs. A representative antigen in this regard is the CD-34 antigen. Representative examples of antibodies which specifically recognize the CD 34 antigen include MY-10 and HPCA2, (Becton-Dickinson, Mountain View, Calif.), QBEND-10 (Quantum Biosystems, Cambridge, U.K.) and 12.8 (CeUpro^®, Bothell, Wash.). This Hgand may be utilized in the above-described devices and methods in order to immobhize the fetal progenitor ceUs. After incubation, the unbound blood products are removed as described above, such that the fetal progenitor ceUs are enriched. Within the context of the present invention, cells are "enriched" if greater than 0.001% of the ceUs are fetal progenitor cells. Preferably, the fetal progenitor ceUs are enriched to greater than .1% and particularly, to greater than 1% of the ceUs present.

Within another aspect of the present invention, a method is provided for enriching fetal progenitor ceUs from maternal blood comprising the steps of (a) incubating a sample of maternal blood with a labeled Hgand capable of specificaUy binding to fetal progenitor cells under conditions and for a time sufficient to aUow specific binding of the Hgand to the ceUs, (b) detecting the presence of the hgand bound cells, and (c) separating the Hgand bound cells from the unbound cells, such that the fetal progenitor cells are enriched. Within this aspect of the invention, ceUs are incubated with a Hgand such as that described above which is capable of specificaUy binding to fetal progenitor cells, except that rather than being immobilized, this Hgand is labeled. Various labels may be utilized within the context of the present invention, although fluoroscein- isothocyanate, phycoerythrin, rhodamine isothiocyanate, or other such highly fluorescent molecules are particularly preferred. Through use of flow cytometry (FACS) labeled cells may then be detected, and separated from non-labeled (or a non-ligand bound) cells, such that fetal progenitor ceUs are enriched.

Within one embodiment of the invention, prior to the step of incubating, red blood ceUs may be removed from maternal blood, for example, by running the maternal blood over a FicoU gradient. Within another embodiment, subsequent to the step of removing the unbound blood products, the bound ceUs are incubated in the presence of erythropoietin utilizing culture methods described above.

Within another aspect of the invention, a method is provided for enriching fetal progenitor ceUs from maternal blood, comprising the steps of

(a) incubating a sample of maternal blood with an immobilized Hgand capable of specificaUy binding to cells other than fetal progenitor cells under conditions and for a time sufficient to aUow specific binding of the hgand to the other ceUs, and

(b) removing the nonbound fetal progenitor cells, such that the fetal progenitor cells are enriched. As noted above, CD 34 is a ceU-surface antigen that may be found on fetal progenitor cells. Many ceU-surface antigens however, can not be found on progenitor cells, and thus maybe utilized to deplete a sample of maternal blood from cells other than fetal progenitor ceUs. Representative examples of such antigens include la, Glycophorin, CD 3, CD 19, CD 11, CD 14, CD 33, and CD 45. Ligands, such as antibodies, which specificaUy bind to such antigens may be purchased from commercial supphers such as Becton-Dickinson, Mountain View, Calif.

Within one embodiment of this invention, prior to the step of incubating, red blood cells are removed from maternal blood by, for example, running the maternal blood over a FicoU gradient. USE OF FETAL CELLS

As described herein, enriched fetal ceUs have a variety of uses. For example, through in situ hybridization the presence of a selected genetic material may be detected in a fetal ceU. SimUarly, DNA or RNA amplification may also be used to detect a selected genetic sequence in fetal ceUs. The methods of the present invention are particularly useful for fetal ceUs which are enriched to such an extent that they are amenable to chromosomal typing by conventional cytogenetics techniques wherein spreads of the ceUs' chromosomes are examined under a microscope.

In situ hybridization may be used as a method for detecting the presence of a selected genetic material within cells. See Pinkel et al, Proc. Nail Acad. Set USA 55:9138-42 (1988); see also Hopman et al, "Detection of Numerical Chromosome Aberrations in Bladder Cancer by In Situ Hybridization," Am. J. of Path. i35(6):1105-1117 (1989). Briefly, the genetic material associated with the preferentially enriched ceUs is first exposed using techniques well known in the art. The genetic material is then incubated with a labeled probe capable of specificaUy hybridizing to the genetic material under conditions and for a time sufficient to aUow hybridization to occur. See, for example, K. E. Davies, Human Genetic Diseases, IRL Press, Oxford, U.K. (1986). FinaUy, the presence of the hybridized labeled probe is detected. Within a preferred embodiment, the genetic material is denatured after the step of exposing. Within the context of the present invention, genetic material includes whole chromosomes, DNA and RNA

Various methods are also weU known in the art for amplifying and detecting genetic material. For example, if present, a selected genetic sequence may be amplified using techniques well known in the art, and then probed for presence of that sequence. See Kogan et al, "An Improved Method For Prenatal Diagnosis of Genetic Diseases by Analysis of Amplified DNA Sequences," The New Eng. J. ofMed. 377(16):985-990 (1987); see also Witt and Erickson, "A Rapid Method for Detection of Y-Chromosomal DNA from Dried Blood Specimens by the Polymerase Chain Reaction," Human Genet. 52:271-274 (1989). Methods for amplification include Polymerase Chain Reaction ("PCR") (see MulHs et al, U.S. Patent No. 4,683,195; MulHs et al, U.S. Patent No. 4,683,202; and Mullis et al, U.S. Patent No. 4,800,159, which are incorporated herein by reference), and RNA- based amplification techniques. See Lizardi et al, Bio/Technology 6:1197-1202 (1988); Kramer et al, Nature 339:401-402 (1989); and Lomeli et al, Clinical Chemistry 35(9): 1826-1831 (1989); see also Kramer et al, U.S. Patent No.4,786,600, which is incorporated herein by reference.

PCR is the most commonly used method for amplifying DNA sequences. Briefly, amplification entails adding the appropriate primer(s), enzymes and nucleotides into a reaction mixture, foUowed by several (20-80) cycles of denaturation and annealing in order to amplify the smaU amount of target DNA The DNA mixture is then separated by electrophoresis and hybridized with a labeled probe to detect the presence of the target sequence of DNA The preferentiaUy enriched fetal ceUs may also be chromosomaUy typed. See Human Cytogenetics, D.E. Rooney and B.H. Czepulkouski (eds.), IRL Press, Oxford, U.K. (1986). Briefly, within a preferred embodiment, a sample containing at least 1 fetal ceU in 10^ other cells is cultured for 2-6 days in Iscoves' Modified Dulbecco's Medium (IMDM) (Gibco, Grand Island, N.Y.) containing a final concentration of 20% fetal bovine serum (FBS) (HYCLONE™, Logan, Utah) and 2 U/ml of highly purified recombinant erythropoietin (Terry Fox Laboratory, Vancouver, B.C., Canada) in order to increase the proportion of ceUs in metaphase. The cells are then incubated in colcemid, fixed, attached to microscope shdes, trypsin-treated and stained with Wright's stain. The slides may then be scanned microscopicaUy for abnormal chromosomes.

PreferentiaUy enriched fetal ceUs may also be utilized therapeuticaUy. For example, within one aspect of the invention, a gene may be inserted into a retrovirus, and the retrovirus utilized to infect fetal cells which are deficient in this gene. The infected fetal ceU may then be administered to the fetus (or another individual) in order to remedy the genetic defect. Diseases which may be treated in this manner are numerous, including for example ADA, sickle ceU anemia, Thalassemia, and SCLD. Methods for inserting genes into retroviruses are also weU documented (see, for example, WO 90/01870, WO 88/03167, WO 88/09670, WO 89/07150, WO 89/11539 and WO 89/09271, aU of which are incorporated by reference).

Fetal cells may also be utilized as universal donor cells. Briefly, because fetal progenitor ceUs do not create as strong an immune reaction in recipients as do progenitor cells from adults, they can be transplanted with fewer side effects. These fetal progenitor cells can thus be used for transplant to correct inherited metabohc diseases in recipients. By expanding their numbers in culture, these fetal progenitor cells may even be used in place of a bone marrow transplant foUowing myeloblative therapy for cancer. PreferentiaUy, enriched fetal ceUs may also be made oncogenic in order to study the progress of such oncogenic cells, or in order to simplify expansion and maintenance of fetal cells for research or diagnostic purposes.

Representative transforming genes that may be inserted into fetal ceUs (for example, by retroviruses as discussed above) include SV40, Ti, myc, ras and src.

PreferentiaUy, enriched fetal cells may also be cryopreserved for future research, or for future therapeutic use. Briefly, methods for freezing stem ceUs are described in a pending apphcation entitled "Method for freezing engrafting ceUs" (Attorney's Docket No. 200072.409). These methods may also be utilized to freeze fetal progenitor cells.

The foUowing examples are offered by way of illustration, and not by way of hmitation.

EXAMPLES

EXAMPLE 1 Carboxylation of a Polyacrylamide Gel

Seventeen grams of dry Biogel P-60¹" (50-100 mesh (wet), coarse beads) (BIORAD, Catalog No. 150-1630, Richmond, Cahf.) are added to 1.5 1 of 0.5 M NaHCO3/0.5 M Na2Cθ3. The pH is adjusted to 10.5 with NaOH and carefully stirred with a mixer (RZR1, Carfamo, Wiarton, Ontario, Canada) so as not to damage the beads for approximately 20 to 30 minutes. The mixture is then placed in a 60°C water bath. After the mixture reaches a temperature of 60°C, it is incubated for an additional 2 hours (at 60°C) with occasional stirring. The mixture is then removed from the water bath, and placed in an ice bath to bring the mixture temperature down to room temperature.

The beads are washed several times with distiUed or deionized water, foUowed by several washings of PBS using a coarse glass filter connected to a vacuum. The carboxylated gel may be stored in PBS at 4°C, and is stable for up to one year if sterilized or stored with a preservative. EXAMPLE 2 Avidin Conjugating the Carboxylated Biogel

PBS is first removed from a measured amount of carboxylated Biogel by filtering with a coarse glass filter connected to a vacuum. The gel is then equihbrated in distiUed or deionized water for 15-30 minutes. EquiUbration in water causes an expansion of the gel to a volume of about 4 times its previously measured amount. The gel is resuspended in 10 ml of distiUed or deionized water for each ml of gel (as originaUy measured in PBS). Thirty mg of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide

(EDC-HC1) (Sigma Chemical Co., Catalog No. E7750, St Louis, Mo.) is added for each ml of gel as originaUy measured. The pH is rapidly adjusted to 5.5 by dropwise addition of Hd Care is taken to maintain the pH at 5.5; pHs of less than 5.0 or greater than 6.0 result in significantly less activation of the Biogel. The mixture is stirred for five minutes.

Avidin (International Enzymes, Inc., FaUbrook, Calif.) is dissolved at a concentration of between 10 and 100 mg/ml in deionized water. Next, 1000 μg of avidin is rapidly added for each ml of gel (as originaUy measured in PBS). The mixture is stirred for 1.5 hours. Next, 2 M glycine is added to give a final concentration of 0.2 M glycine in the mixture and stirred for an additional 1 hour. The gel is washed with several volumes of PBS using a coarse glass filter and vacuum, and stored in PBS at 4°C. The gel is stable for approximately one year.

EXAMPLE 3

-tomunoadsorption of Maternal CeUs With The Two-Step Method

PREPARATION OF CELLS Twenty rnillihters of blood is obtained from a pregnant female and suspended in an equal volume of PBS with 1% Bovine Serum Albumin (BSA) (Sigma Chemical Co., St. Louis, Mo.) in four 50 ml centrifuge tubes. The number of nucleated cells/ml is counted in the foUowing manner: A 50 μl aliquot of mixed, anticoagulated whole blood is dUuted into 3 ml of a 3% acetic acid solution. After vortexiπg, 7 μl samples of diluted blood are loaded into each of two chambers of a hemacytometer. After about 3 minutes to aUow settling of the cells, the nuclei of ceUs, which are almost aU lysed by the acetic acid, are counted, in the four ruled fields per chamber of a hemacytometer with improved Neubauer ruHngs (VWR Scientific, San Francisco, Calif.), each field representing 0.1 x 10^"^ μl sample volume. The average number of nuclei per field is multiplied by a dUution factor of 61 x 10⁴ to calculate the number of nucleated ceUs per miUihter of whole blood. If the average is less than 10 nuclei per field, the procedure is repeated, except 50 μl blood is dUuted into only 1 ml acetic acid, with a resulting new dUution factor of 21 x 10 .

Each tube of diluted blood is underlayered with 5 ml of Histopaque 1077 (Sigma Chemical Co.) and centrifuged at 700 x g for 15 minutes at room temperature. CeUs at the interface are coUected and washed once in PBS plus 1% BSA The pellet is resuspended in 100 μl in PBS plus 1% BSA

Twenty micrograms/miUiliters of anti-transferrin receptor antibody

(Becton Dickinson, Immunocytometry Systems, Mountain View, Calif.) is added to the mixture 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 5 minutes.

The ceUs are then gently resuspended to 1 ml and 1 μg/ml of biotinylated goat anti-mouse IgG (Southern Biotech, Birmingham, Ala.) is added. The mixture is incubated for 15 minutes on ice and washed twice as described above with PBS plus 1% BSA

PREPARATION OF COLUMNS

Carboxylated Biogel P-30™ (prepared as described above) is allowed to equihbrate to room temperature and placed in a K9/15 column (Pharmacia,

Piscataway, NJ.) to a total bed height of 1 cm. The column is washed with PBS, followed by washes with PBS plus 5% BSA. This column functions as a "pre- column." The avidin column contains avidin-conjugated Biogel P-60™, which is prepared as described above. The avidin-conjugated Biogel is allowed to equUibrate to room temperature and placed in a K9/15 column to a total bed height of 4 cm. The column is then washed with several volumes of PBS, followed by washes with PBS plus 5% BSA

IMMUNOADSORPTION OF CELLS

CeUs which have been prepared as described above are resuspended in PBS plus 5% BSA to a volume of 1 ml. The ceUs are then gently transferred onto the top of the gel bed of the pre-column filter. The ceUs are allowed to filter through the pre-column and are washed with 1 ml PBS plus 5% BSA A peristaltic pump (Cole-Parmer, Rockford, HI.) controls flow from the avidin column to a rate of about 1 ml/minute. Once the ceUs have almost run down to the top of the avidin column bed, 1-2 ml of PBS plus 5% BSA is added to the top of the avidin column in order to wash out remaining cells. The column is washed out with 4-6 ml of PBS plus 5% BSA, foUowed by 4-6 ml of PBS.

REMOVAL OF ADSORBED CELLS FROM THE AVΓDIN COLUMN The avidin column 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 pipette is used for mechanicaUy agitating and resuspending the ceU bed, thus aUowing ceUs to become detached from the gel matrix, and to filter into the centrifuge tube. The tube is then centrifuged at 400 x g for 5 minutes and resuspended in ceU culture media as described below.

EXAMPLE 4

Preferential Enrichment of Fetal Nucleated Erythroid Cells

ENRICHMENT OF FETAL CELLS WITH ERYTHROPOIETIN

Cells which are separated from the Avidin-Biogel column above, are resuspended in Iscoves' Modified Dulbecco's Medium (IMDM) (Gibco, Grand

Island, N.Y.) containing a final concentration of 20% fetal bovine serum (FBS)

(HYCLONE¹", Logan, Utah), and 2 U/ml of highly purified recombinant erythropoietin (Terry Fox Laboratory, Vancouver, B.C., Canada). CeUs are dUuted to 5 x 10" nucleated cells/ml and 200 μl is plated into each weU of a 96 weU tissue culture plate with round bottoms (Coπiing Glass Works, Corning,

N.Y.).

ENRICHMENT OF FETAL CELLS BY AMMONIUM ION DIFFERENΠATION

CeUs which are separated from the Avidin column above are adjusted to a concentration of less than 2 x 10 ' nucleated ceUs/ml. One volume of the ceU mixture is chUled, and a solution at 29°C containing 18 volumes of 0.1844 M NH4CI and 2 volumes of 10 μM acetazolamide is added. After 2 minutes, 2 volumes of 3mM NH4HCO3 is rapidly added, and the whole mixture gently stirred for 3 minutes. CeUs are washed several times with PBS by centrifugation in order to remove ceUular debris. EXAMPLE 5 In Situ Hybridization

Enriched ceUs are exposed to a hypotonic solution (.075 M KC1) for 12 minutes at 37°C. The tubes are inverted once during the incubation to keep the ceUs suspended. Twenty drops of freshly prepared fixative (3:1 methanolracetic acid) is added to the ceUs, vortexed, and then centrifuged for 8 minutes at approximately 250 x g. Fresh fixative is added to the ceUs, foUowed by incubation for one hour at room temperature. The cells are centrifuged for 8 minutes at approximately 250 x g. Fresh fixative is added and the process is repeated one more time. FinaUy, the ceUs are resuspended in a small amount of fixative and placed at 4°C overnight.

The next day the ceUs are vortexed and placed onto microscope shdes (Baxter, McGaw Park, 111.) which have been cleaned with ethanol and dipped in distilled water. The shdes are aUowed to dry for two days at room temperature.

The slides are heated in 70% formamide in 2 x SCC (0.30 M NaCl,

0.030 M Na citrate) to 68°C-70°C for 2.5 minutes. The shdes are then immediately placed in a rinse of 70% ethanol in water. FoUowing the rinse, the shdes are placed successively in 70%, 95%, and 100% ethanol solutions for 5 minutes each.

Each alcohol solution must be maintained at < -20°C. The shdes are air dried.

A probe is prepared from plasmid DNA according to the method of Page et al ("Single copy sequence hybridizes to polymorphic and homologous loci on human X and Y chromosomes," PNAS 79:5352-5356 (1982), from American Type Culture Collection (ATCC) No. 57261; except that biotin-dATP is incorporated into the probe. Twenty five microhters of the biotinylated probe (5 μg/ml) per slide is placed in a microfuge tube. The probe is heated to 70°C for 5 minutes then immediately placed on ice. Twenty microhters of the probe solution is placed onto each slide and covered with a 22 x 40 mm covershp. The shdes are placed into a box with a wet paper toweling liner and incubated at 37°C for 12-18 hours.

A 50% solution of formamide in 1 x SCC is warmed to 37°C. Covershps are removed from the shdes and immersed into the 50% formamide solution for 30 minutes. The shdes are then placed in 2 x SCC solution for 30 minutes with gentle rocking, then in 1 x SCC for 30 minutes with gentle rocking. Fluoresceinated avidin (Vector, Burlingame, Calif.) is dUuted 1:1000 (1 μg/ml). The back of the slide and around cell area are wiped. Two hundred microhters of the avidin-fluorescein is added to each slide and incubated in the box for 30 minutes at room temperature. The sHdes are rinsed sequentially in (1) 4 x SCC for 10 minutes with rocking, (2) 4 x SCC, 0.1% Tween-20 for 10 rninutes without rocking, and (3) 4 x SSC for 10 minutes with rocking. The back of the slide and around ceU area is wiped off. Ten microhters of anti-fade plus propidium iodine (10 ml PBS, 100 mg p-Phenylene diamine, 90 ml glycerol, pH 8.0, 10 μg/ml propidium iodide) is added. The shdes are covered with coversHps and stored at 4°C. The shdes may be stored for several days if necessary. Target DNA may be. observed under a microscope by the presence of fluorescence.

EXAMPLE 6 Chromosomal Typing

The enriched ceUs are exposed to 1 μg of colcemid (Sigma, St. Louis, Mo.) for one hour at 37°C. A hypotonic solution (0.075 M KC1) is added to the ceUs and incubated for 12 minutes at 37°C. The tubes are inverted once during the incubation to keep the cells suspended. Twenty drops of freshly prepared fixative (3:1 methano acetic acid) is added to the cells, vortexed, and then centrifuged for 8 minutes at approximately 250 x g. Fresh fixative is added to the cells, foUowed by incubation for 1 hour at room temperature. The cells are centrifuged for 8 rninutes at approximately 250 x g and fresh fixative is added again. This process is repeated one more time. FinaUy, the ceUs are resuspended in a smaU amount of fixative and placed at 4°C overnight. The next day the ceUs are vortexed and placed onto microscope shdes (Baxter, McGaw Park, LI.) which have been cleaned with ethanol and dipped in distilled water. The shdes are aUowed to air dry for about 3-4 days, and then are treated with 0.005% trypsin (Difco Bactotrypsin, VWR Scientific, San Francisco, Cahf.) for 30 to 35 seconds. The shdes are washed twice in PBS Plus 1% FBS, foUowed by washing in PBS only. The cells are stained with Wright's solution (Sigma Chemical Co., St. Louis, Mo.), foUowed by two washes with deionized water. The shdes are scanned for evidence of metaphase cells and typed by conventional cytogenetics. EXAMPLE 7

Culture Without Separation

A MATERNAL BLOOD Maternal samples were dUuted 1:1 with Phosphate Buffered Saline

(PBS). Twenty miUihters were placed in a centrifuge tube, foUowed by 8 ml of FicoU Hypaque. The tube was centrifuge for 15 minutes at 1700 rpm (500xg). The peUeted ceUs were washed twice by resuspending and then centrifuging the ceUs. The ceUs were resuspended in 20 ml of IMDM culture media containing 20% Fetal Bovine Serum (HYCLONE, Logan, Utah), 2mM glutamine, ImM sodium pyruvate, 0.1 mM NEAA (non-essential amino acids; Whittaker Bioproducts, WalkersvUle, Md.), and 1 U/ml EPO (Terry Fox Laboratory, Vancouver, B.C., Canada. Ten miUihters of the ceU suspension was placed into each of two T75 Costar flasks, and incubated overnight at 37°C in a 5% CO2 incubator. Adherent ceUs such as fibroblasts are thus removed prior to separation.

EXAMPLE 8 Separation of Fetal Progenitor CeUs on an Affinity Column

Samples from four patients were treated as described above in

Example 7. After incubation overnight, the cell culture was counted and centrifuges 10 minutes at 1200 rpm. The supernatant was removed, and the ceUs were resuspended in 10 ml of PBS containing 1% BSA (Bovine Serum Albumin) and centrifuged once more for 10 minutes at 1200 φm (250 x g). The supernatant was removed and the ceUs were resuspended in 1 ml of PBS containing 0.1% BSA

Antibody 12.8 (CellPro, BotheU, Wash.) (an anti-CD 34 antibody) was added to the ceU suspension to a final concentration of 40 ug/ml. The ceUs were then incubated for 30 minutes on ice. After 30 rninutes, the cells were washed twice by centrifugation and resuspension in PBS/1% BSA Next, biotinylated rabbit anti-mouse IgM antisera

(Zymed Laboratories, South San Francisco, Cal.) was added to a final concentration of 1:1000 and incubated for 30 minutes on ice. The cells were washed twice by centrifugation and resuspension in PBS/1% BSA, and finally resuspended in 1 ml of PBS containing 5% BSA

An avidinated gel was prepared essentially as described in Examples 1 and 2 above, and placed into a soft column (see pending apphcation U.S. Serial No. 07/599,796, which is hereby incoφorated by reference) to a bed depth of 4 cm.

EXAMPLE 9 Culture Post Separation

Adsorbed cells which were separated by the above procedure were resuspended in IMDM containing 20% FBS and 2 U/ml EPO. The cells were- then placed in 96 weU Costar plates in a volume of 200 μl per weU (approximately one million cells per weU), and incubated for 5 days at 37°C in a 5% CO2 incubator.

A PREPARATION OF CELLS FOR METAPHASE SPREADS

After incubating for five days, the ceUs were transferred into nήcrocentrifuge tubes and exposed to Colcemid (1 μg in 300 μl) for one hour at 37°C. The cells were then centrifuged for 5 minutes at 1000 rpm, and 1 ml of 0.075M KQ was added. Twenty drops of freshly prepared fixative (3:1 MethanofcAcetic Acid) was added to the ceU suspension, and the ceUs were centrifuged again for 5 minutes at 1000 φm. One ml of fixative was added and the cells were left at room temperature for 1 hour. The cells were then centrifuged for 5 minutes at 1000 φm, and washed three times in 1 ml of fixative, and left in 500 μl overnight at 4°C. The next day metaphase spreads were prepared.

B. METAPHASE SPREADS (Karotyping)

The spreads were aUowed to dry several days at room temperature. Next, the shdes were placed in Difco Bactotrypsin for one minute (6 drops/45 ml PBS). The shdes were rinsed in PBS containing 1% FBS, and then in PBS only. The shdes were placed in fresh Wright's stain for 35 seconds, rinsed twice, and scanned under the microscope.

EXAMPLE 10 Determination of CFCs

One ml per 35 mm plate of Iscove's MethylceUulose (Terry Fox

Laboratories, Vancouver, British Columbia, Canada) supplemented with 2 mM L- glutamine and 50 μg/ml gentamicin was warmed to 37°C. CeUs were plated in triplicate at 3-fold dilutions to improve the accuracy of the assay. The highest number of cells plated was 10⁵/plate except for column-purified ceUs which were plated at 3 x 10³ and less. The ceUs were spread evenly over the surface of each plate and then incubated in a humidified incubator at 37°C with 5% CO2 in air for 10 to 14 days. Colonies were counted if they contained more than 50 cells and scored as CFU-GM, BFU-E, or other (e.g., CFU-GEMM). The number of various types of colonies were summed to give the total number of colony-forming ceUs (CFC).

The results are summarized briefly in Table 1 below:

TABLE 1

When CFCs are compared, it is evident that a significantly higher number of ceUs were obtained from the pregnant female, as compared to the non- Pregnant female or the male. Although it is possible that the increase in number is due to mobilization of progenitor cells from the pregnant female's bone marrow, at least a portion of the increase in progenitor ceUs is due to fetal progenitor ceUs in the mother's circulation. EXAMPLE 11 In Situ Hybridization

Fetal ceUs which had been enriched as described above in Example 8 were subjected to in situ hybridization utilizing a commerciaUy avaUable kit (Chromosome in situ kit S1370, Oncor, Gaithersburg, MD). CFCs number was also determined as described above in Example 10. The results are briefly set forth below in Table 2.

TABLE2

SELECTED RESULTS OF FETAL CELL SEPARATION FROM MATERNAL BLOOD FOLLOWED BY CULTURE AND IN SITU HYBRIDIZATION

WΓΓHY-PROBE

EXAMPLE 12 FACS Analysis

Fetal cells which were enriched as described above in Example 8 were submitted to analysis by FACS. Briefly, approximately 125,000 purified ceUs were divided into two tubes. One tube received an IgG control, and the second received QBend-10 (an anti-CD 34 antibody) at a final concentration of 20 μg/ml. The tubes were incubated for 30 minutes on ice, then washed twice with 4 ml of 1% BSA in PBS.

Both tubes were then treated with a 1:50 dUution of [FITC- conjugated] 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 ceUs were suspended in 200 μl of PBS and propidium iodide (1 μg/ml) and analyzed on a FACScan (Becton Dickinson). EXAMPLE 13 Plucking and Reculturing Colonies

Individual fetal ceU colonies were sterilely plucked from the methylceUulose culture of Example 10. The colonies were placed into Ex Vivo media, and then placed back into methylceUulose culture to observe differentiation. From the single ceU, a colony of clonal fetal cells develops.

Clonal fetal cells are identical, and thus may be utilized for assays wherein the effect of multiple samples are to be tested on ceUs (ie., to establish whether a compound is carcinogenic). Some of the ceUs may function as a control, whUe other ceUs may be subjected to the compound. Similarly, multiple assays may be performed in order to determine the sensitivity of a ceU to a certain drug. In addition, the colonies may be screened for a desired response, and clones subjected to more detailed analyses.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for the puφoses of iUustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

Claims

1. A method for enriching fetal progenitor cells from maternal blood, comprising: 5 (a) incubating a sample of maternal blood with an immobilized

Hgand capable of binding to fetal progemtor cells under conditions, and for a time . sufficient to aHow specific binding of said Hgand to said cells; and

(b) removing unbound blood products such that said fetal 0 progenitor cells are enriched.

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

_ 5 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 ⁰ eiythropoietin.

5. A method for enriching fetal progenitor cells from maternal blood, comprising:

(a) incubating a sample of maternal blood with a labeled hgand 5 capable of binding to fetal progemtor ceUs under conditions and for a time sufficient to aHow specific binding of said ligand to said ceils;

(b) detecting the presence of said Hgand bound cells; and

(c) separating said Hgand bound ceils from the unbound cells, such that said fetal progenitor cells are enriched. 0

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 5 separating the maternal blood on a density gradient. 31

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 eiythropoietin.

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

10. The method of claims 1 or 5 wherein said Hgand is an antibody.

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

12. A method for enriching fetal progemtor cells from maternal blood, comprising: (a) incubating a sample of maternal blood with an immobilized

Hgand capable of binding to cells other than fetal progenitor ceils under conditions and for a time sufficient to allow specific binding of said Hgand to said other cells; and

(b) removing the nonbound fetal progenitor ceUs, such that said fetal progenitor cells are enriched.

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

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

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

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

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.

SUBSTITUTE SHEET

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

SUBSTITUTE SHEE