WO1995026417A1 - Culture et isolation de cellules f×tales a partir de sang peripherique maternel - Google Patents

Culture et isolation de cellules f×tales a partir de sang peripherique maternel Download PDF

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Publication number
WO1995026417A1
WO1995026417A1 PCT/US1995/003906 US9503906W WO9526417A1 WO 1995026417 A1 WO1995026417 A1 WO 1995026417A1 US 9503906 W US9503906 W US 9503906W WO 9526417 A1 WO9526417 A1 WO 9526417A1
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cells
fetal
sample
erythroid
peripheral blood
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PCT/US1995/003906
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English (en)
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Karen Pavelka
Anna Mahr
Katherine W. Klinger
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Genzyme Corporation
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Priority to EP95916136A priority Critical patent/EP0804614A1/fr
Priority to JP7525275A priority patent/JPH09510875A/ja
Priority to AU22745/95A priority patent/AU2274595A/en
Publication of WO1995026417A1 publication Critical patent/WO1995026417A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0641—Erythrocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/14—Erythropoietin [EPO]

Definitions

  • All hematopoietic cells are derived from pluripotent stem cells which, under appropriate conditions, differentiate along one of several possible hematopoietic lineages.
  • One of these lineages, the erythroid lineage leads to the production of red blood cells.
  • the development of mature, non-nucleated red blood cells, or erythrocytes results from the commitment of pluripotent stem cells to the erythroid lineage, where they become erythroid progenitor cells, and further differentiation of erythroid progenitor cells into mature cells in response to signals received by the developing cells.
  • An early erythroid progenitor cell is the blast forming unit-erythroid (hereafter BFU-E), which develops into a more mature erythroid progenitor cell, the colony forming unit-erythroid (hereafter CFU-E).
  • BFU-E blast forming unit-erythroid
  • CFU-Es colony forming unit-erythroid
  • nRBCs nucleated red blood cells
  • erythroid progenitor cells are located predominantly in the bone marrow, where hematopoiesis occurs in adults, and the erythroid progenitor cells do not circulate in peripheral blood.
  • the protein erythropoietin is a growth factor necessary for erythroid development in vivo which is produced primarily by the kidneys. Erythropoietin was originally isolated and purified from urine of anemic patients. Miyake, T., et al. J. Biol Chem. 252, 5558-5564 (1977). More recently, erythropoietin has been molecularly cloned and characterized. Jacobs, K., et al., Nature 313, 806-809 (1985); Lin, P., et al., Proc. Natl. Acad. Sci. USA 82, 7580-7584 (1985).
  • Erythropoietin has been used to support the growth of erythroid progenitor cells in culture in vitro.
  • human adult erythroid progenitors from bone marrow and fetal erythroid progenitor cells from umbilical cord blood or fetal liver explants have been grown in vitro by culturing cells in media containing erythropoietin.
  • fetal cells are present in maternal blood in very limited numbers, which requires that they be enriched within a mixture of fetal and maternal cells or that the fetal cells be separated in some way from maternal cells.
  • One approach that has been used to achieve enrichment or separation of fetal cells utilizes antibodies specific for a particular fetal cell type.
  • fetal-specific antibodies can be used in order to facilitate separation of fetal cells from maternal components by flow cytometry.
  • the present invention provides non-invasive methods for culture and isolation of fetal cells and for detecting nucleic acid sequences of interest in isolated fetal cells.
  • the invention is based, at least in part, on the discovery that fetal cell, e.g., fetal progenitor cells, are present in the peripheral blood of a pregnant woman and can be selectively grown in vitro by culturing the cells in the presence of a cell growth factor.
  • the invention provides non-invasive methods for culture and isolation of fetal erythroid cells and for detecting nucleic acid sequences of interest in isolated fetal erythroid cells.
  • the invention is based, at least in part, on the discovery that fetal erythroid progenitor cells are present in the peripheral blood of a pregnant woman and can be selectively grown in vitro by culturing the cells in the presence of an erythroid growth factor, e.g., erythropoietin.
  • the methods of the invention offer a non-invasive means by which to obtain fetal cells in numbers great enough to be of use diagnostically while not requiring specialized reagents such as monoclonal antibodies or expensive equipment such as a flow cytometer.
  • fetal erythroid progenitor cells in the presence of an erythroid growth factor, e.g., erythropoietin, causes the cells to proliferate and differentiate into fetal erythroid cells. Fetal erythroid cells can then be isolated and used in further analyses.
  • the invention provides methods which allow for enrichment of fetal cells relative to maternal cells in a peripheral blood sample, expansion of the total number of fetal cells present compared to the total numbers present in the original sample, and isolation of mitotically active fetal cells.
  • the ability to culture fetal erythroid progenitor cells from maternal peripheral blood is of great value since this approach provides a simple, non-invasive means of obtaining fetal cells for further analysis.
  • the advantages of this approach are that it allows for isolation of greater numbers of fetal cells for analysis than can easily or economically be separated from maternal blood by other methods and, since these cells are growing, also allows for isolation of fetal cells in metaphase for use in diagnostic tests.
  • this invention pertains to the culture and isolation of fetal erythroid cells from a maternal peripheral blood sample as a means of obtaining fetal cells for analysis and diagnosis.
  • the method of the invention involves obtaining a sample of maternal peripheral blood, culturing cells in the sample in a culture medium containing an erythroid growth factor, e.g., erythropoietin, which causes erythroid progenitor cells to proliferate and differentiate into erythroid cells, and isolating fetal erythroid cells.
  • an erythroid growth factor e.g., erythropoietin
  • This method is based at least upon: 1) the presence of fetal erythroid progenitor cells in the maternal peripheral circulation; 2) the scarcity of maternal erythroid progenitor cells in the maternal peripheral circulation; and 3) the higher sensitivity of fetal erythroid progenitors to erythropoietin compared to the sensitivity of maternal erythroid progenitor cells to erythropoietin.
  • the peripheral blood sample Prior to culturing, the peripheral blood sample can be treated in order to remove certain cell types and/or to enrich for certain cell types so that fewer total cell numbers are utilized in the culturing step.
  • Non-nucleated red blood cells can be removed from the sample by, for example, selective lysis or density gradient centrifugation prior to culturing.
  • Erythroid progenitor cells can be enriched in the sample by, for example, performing dual density gradient centrifugations, wherein gradients of different densities are utilized, and isolating a cell layer which contains erythroid progenitor cells prior to culturing.
  • erythroid cells can be isolated by picking one or more discrete colonies of cells from the culture media, for example by using a pipette to manipulate the cells. The cells of the colony can then be transferred to fresh culture medium, placed on a microscope slide for further analysis, used as a source of DNA, or analyzed further in any suitable manner.
  • Another aspect of the invention relates to a method for identifying fetal erythroid cells after culturing cells of a maternal peripheral blood sample in an erythroid growth factor, e.g., erythropoietin.
  • This method involves detecting a fetal cell marker on erythroid cells as a means of identifying the cell as being of fetal origin.
  • a further aspect of the invention involves detection of a nucleic acid sequence of interest in fetal nucleic acid of fetal erythroid cells after culturing. Detection of Y chromosomal DNA, a gene associated with a disease-causing mutation or chromosomal abnormalities in DNA from cultured erythroid cells are all within the scope of the invention.
  • the invention also provides a method for isolating fetal cells in metaphase from a maternal blood sample.
  • a method for isolating fetal cells in metaphase from a maternal blood sample.
  • cells are exposed to an agent which inhibits progression of dividing cells through the cell cycle or an agent which synchronizes growth of cells.
  • Cells in metaphase can be detected microscopically and isolated. Metaphase cells can then be used for further analysis and diagnostic tests.
  • the invention still further provides a method for preferentially isolating fetal cells from a current pregnancy in a woman who has had multiple pregnancies by culture of fetal erythroid progenitor cells from a peripheral blood sample obtained from the pregnant woman.
  • This method is based upon the short life span, about 3 months, of erythroid progenitor cells such that any fetal erythroid cells that are isolated after culturing must be derived from the current fetus rather than any previous fetus.
  • Figure 1 is a schematic diagram illustrating the relative distribution of erythroid progenitor cells and nucleated red blood cells along a density gradient.
  • Figure 2 is a photograph of a CFU-E colony growing in methylcellulose medium containing erythropoietin.
  • Figure 3 is a photograph of an early BFU-E colony growing in methylcellulose medium containing erythropoietin.
  • the present invention pertains to methods of culturing fetal cells from a sample of maternal peripheral blood.
  • the methods include obtaining a sample of peripheral blood from a pregnant woman and culturing cells within the sample in a culture medium containing a cell growth factor. Fetal cells are isolated from the culture medium and a nucleic acid sequence of interest is detected in the fetal DNA.
  • the invention pertains to methods of culturing fetal erythroid cells from a sample of maternal peripheral blood.
  • the methods involve culturing fetal erythroid progenitor cells present in the blood sample in vitro in a medium containing an erythroid cell growth factor, e.g., erythropoietin, which causes the fetal erythroid progenitor cells to proliferate and differentiate into fetal erythroid cells. Following culture, fetal erythroid cells can be isolated.
  • the method of the invention comprises obtaining a sample of peripheral blood from a pregnant woman, culturing cells within the sample of peripheral blood in a culture medium containing an erythroid growth factor, e.g., erythropoietin, and isolating fetal erythroid cells from the culture medium.
  • a sample of peripheral blood from a pregnant woman also referred to herein as a maternal blood sample
  • a blood sample drawn (e.g. with a needle) from a peripheral blood source e.g. an arm vein
  • the sample of peripheral blood can be treated with an agent which inhibits blood coagulation, such as heparin.
  • the sample of peripheral blood contains approximately 5-20 mis of blood. More preferably, the sample contains approximately 10 mis of blood.
  • the sample may be obtained as early as 6 weeks of gestation or as late as mid-second trimester, but preferably is obtained between about 8 and about 16 weeks of gestation.
  • the sample is obtained at approximately 12 weeks of gestation. It is known that at 12 weeks of gestation there are about 50,000 nRBCs per milliliter circulating in fetal blood whereas by 20 weeks of gestation there are only about 1000 nRBCs per milliliter of fetal blood. See Holzgreve,W., et al. The Journal of Reproductive Medicine 37, 410-418 (1992).
  • culture medium is intended to refer generally to contacting cells with a culture medium appropriate for the survival of the cells and incubating the cells in the culture medium under conditions appropriate for the survival of the cells for a period of time to allow proliferation and possibly differentiation of the cells.
  • culture medium is intended to include a liquid or semisolid solution or material which allows survival and growth of fetal cells, e.g., erythroid progenitor cells.
  • fetal cells e.g., erythroid progenitor cells.
  • Suitable culture media generally contain a nutrient source, such as carbohydrates (e.g. sugars), general growth factors, such as those found in blood serum (e.g. fetal bovine serum), and supplementary amino acid(s), such as L-glutamine.
  • a culture medium can also contain antibiotics, such as penicillin and streptomycin, a reducing agent (e.g. 2-mercaptoethanol), additional protein (e.g. bovine serum albumin), a buffering agent (e.g. sodium bicarbonate) and/or a pH indicator (e.g. phenol red).
  • antibiotics such as penicillin and streptomycin, a reducing agent (e.g. 2-mercaptoethanol), additional protein (e.g. bovine serum albumin), a buffering agent (e.g. sodium bicarbonate) and/or a pH indicator (e.g. phenol red).
  • antibiotics such as penicillin and streptomycin, a reducing agent (e.g. 2-mercaptoethanol), additional protein (e.g. bovine serum albumin), a buffering agent (e.g. sodium bicarbonate) and/or a pH indicator (e.g. phenol red).
  • Conditions appropriate for survival of mammalian cells in culture generally are 37 C, 4 %-5 % CO2.
  • the culture medium further contains a semisolid matrix material.
  • semisolid matrix material is intended to include a substance which, when added to a liquid culture medium, can convert the liquid culture medium to a gelatinous, semisolid state.
  • a semisolid medium is one in which cells can still grow (i.e. which is not so solid as to inhibit cell growth) but which is firm enough so that isolated cells within the medium cannot migrate from their site of growth.
  • a semisolid matrix material can also allow growing cells to attach to the matrix material, thereby preventing their migration within the medium. Growth of a single cell in a semisolid medium results in the production of a discrete colony of cells derived, by division, from the original single cell.
  • a cell colony can be distinguished from other cell colonies by light microscopic examination of the semisolid culture medium.
  • Suitable semisolid matrix materials include methylcellulose, agargel and a plasma clot. See for example Stephenson, J.R., et al. Proc. Natl. Acad. Sci. USA 68, 1542-1546 (1971); Iscove, N.N., et al. J. Cell Physiol. 83, 309-320 (1974); and Pike. B.L. and Robinson, W.A. J. Cell Physiol. 16, 11 (1970).
  • a preferred culture medium for isolation of fetal erythroid cells from a maternal blood sample is Iscove's Modified Dulbecco's Medium (IMDM; commercially available; Sigma, Whitaker) with added 1.1 % methylcellulose, 30 % fetal calf serum, 1 % BSA, 1 % penecillin-streptomycin, 1 % L-glutamine, 10 ⁇ 4 M 2-mercaptoethanol.
  • Preferred culture conditions are incubation of cells at 37 C, 4 %-5 % CO2 for approximately 96 hours.
  • Cells are typically cultured in an appropriate container for the culture medium, such as a culture dish or culture flask.
  • the culture medium in which the cells of the maternal blood sample are cultured also contains a cell growth factor.
  • the language "cell growth factor” is intended to include those factors which stimulate proliferation or differentiation of fetal cells.
  • cell growth factors include: factors which stimulate differentiation of lymphoid progenitor cells, e.g., interleukin-2 (IL-2), interleukin-4 (IL-4),and interleukin-7, and factors which stimulate hematopoietic progenitor cells, e.g., hematopoietic cell growth factors (including erythroid growth factors), e.g., interleukin -3 (IL-3), granulocyte-macrophage colony- stimulating factor (GM-CSF), monocyte-macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), and human recombinant stem cell factor (rhSCF).
  • IL-2 inter
  • the culture medium in which the cells of the maternal blood sample are cultured also can contain an erythroid growth factor.
  • erythroid growth factor is intended to include J .hose factors which support growth and proliferation of human erythroid progenitor cells. Examples include: erythropoietin (EPO), and burst promoting activity (BPA). These erythroid growth factors can be administered alone or in combination with each other.
  • erythropoietin is intended to include a preparation of the protein erythropoietin which supports growth and proliferation of human erythroid progenitor cells.
  • Erythropoietin may be prepared by purification of the protein from a natural source or may be prepared by expression of the erythropoietin gene by recombinant DNA technology.
  • a crude preparation of erythropoietin isolated from human urine can be used and is commercially available from Sigma (catalogue # E5011, 50 U/mg).
  • the erythropoietin is of human origin, although erythropoietin from another species which is capable of supporting the proliferation and differentiation of human erythroid progenitor cells can also be used. Erythropoietin has been shown to function across species. For example, human and mouse cells have both been stimulated by sheep or human erythropoietin (see Iscove, N.N. et al. J.
  • a concentration of erythropoietin is used which is sufficient to achieve the desired result of proliferation and/or differentiation of erythroid progenitor cells.
  • a preferred concentration range for erythropoietin in the culture medium is about 0.25 U/ml to 1 U/ml.
  • Erythroid progenitor cells exposed to erythropoietin in culture will be stimulated to proliferate and differentiate. Erythropoietin preferentially stimulates the proliferation and differentiation of fetal erythroid progenitor cells in cells from a maternal blood sample for a number of reasons.
  • fetal erythroid progenitor cells from human fetal liver are more sensitive to the stimulatory effects of erythropoietin than are erythroid progenitor cells from human adults (see Peschle, C, et al. Blood 58, 565-572 (1981)).
  • concentration of erythropoietin used e.g., 0.25 U/ml to 1 U/ml
  • the optimal concentration for stimulation of adult erythroid progenitors which is 2-4 U/ml (see Linch et al. Blood 59, 976-979 (1982)).
  • erythroid progenitor cells in human adults are found almost exclusively in the bone marrow, the site of adult hematopoiesis, and therefore few, if any, maternal erythroid progenitor cells should be present in maternal peripheral blood.
  • BFU-E fetal erythroid progenitor cells
  • CFU-E fetal erythroid progenitor cells
  • M-BFU-E the mature, or late
  • CFU-E the CFU-E.
  • Colonies derived from M-BFU-Es and CFU-Es can be distinguished from each other and from other cell types by their morphology, which can be determined microscopically.
  • BFU- E-derived colonies usually contain over 100 cells, and can contain several thousand cells, and are arranged in a diffuse "burst" or scattered pattern of cells.
  • CFU-E-derived colonies contain about 20 to 150 cells arranged in a smaller, more compact colony.
  • CFU-E-derived colonies mature during the first week of culture, whereas BFU-E-derived colonies may take longer to develop. Additionally, CFU-E-derived colonies may undergo further maturation and differentiation to form erythroid cells such as nucleated erythroblasts, which display visible hemoglobinization, which can be identified microscopically.
  • erythroid cells After culturing cells in a culture medium containing an erythroid growth factor, e.g., erythropoietin, erythroid cells are isolated from the culture medium.
  • isolated is intended to refer to separation of one or more cells or colonies of cells away from other cells and/or away from the culture medium.
  • cells are grown in a semisolid culture medium at low enough cell density such that discrete, distinguishable cell colonies form, these cell colonies are already isolated from other cells within the semisolid medium.
  • Cells or cell colonies can be further isolated by removing the cells from the medium. For example, a discrete cell colony can be identified by examining the cell culture under a light microscope and can be removed from the culture medium by picking out the cells from the culture medium.
  • Cells can be effectively picked out of the culture medium using an instrument, such as a drawn out Pasteur pipette or other suitable pipette, to manipulate the cells.
  • an instrument such as a drawn out Pasteur pipette or other suitable pipette, to manipulate the cells.
  • a single colony of cells is picked from the culture medium and transferred to a microscope slide.
  • the language "fetal cells” is intended to include those fetal cells present in the maternal peripheral blood circulation.
  • the fetal cells include fetal progenitor cells, which after incubation with a cell growth factor, can differentiate into mature cells, e.g., granulocytes, monocytes or leukocytes.
  • fetal lymphoid progenitor cells can be cultured with IL-2, IL-7, or IL-4 in order to differentiate into mature T or B-lymphocytes.
  • Other fetal progenitor cells can be cultured with factors such as G-CSF and /or GM-CSF in order to differentiate into mature granulocytes.
  • erythroid cells is intended to include those cells which are derived from erythroid progenitor cells upon culture with an erythroid growth factor, e.g., erythropoietin.
  • Culture of erythroid progenitor cells with an erythroid growth factor, e.g., erythropoietin can cause progenitor cells to differentiate into a more mature stage of erythroid lineage development.
  • the cells present after culture may be a mixture of cell types at different stages of the erythroid developmental pathway. Cells at different stages of the erythroid developmental pathway can be morphologically different from each other.
  • Cells or colonies of cells having a morphology of erythroid progenitor cells, such as BFU-E and CFU-E colonies, or more developed erythroid cells, including cells with visible hemoglobinization, such as nucleated erythroblasts, are detectable after culture of erythroid progenitor cells in an erythroid growth factor, e.g., erythropoietin, and can be isolated from the culture medium.
  • an erythroid growth factor e.g., erythropoietin
  • the sample Before culturing cells from a sample of peripheral blood obtained from a pregnant woman, the sample can be manipulated to enrich for one or more types of cells within the sample.
  • a maternal blood sample contains both nucleated cells, such as lymphocytes, monocytes, granulocytes and, potentially, fetal cells, e.g., fetal erythroid progenitor cells, and non-nucleated mature red blood cells.
  • Non-nucleated mature red blood cells constitute the vast majority of cells within a sample of peripheral blood and it is often desirable to remove these cells from the sample before culturing.
  • a proportion of nucleated cells present in the sample of peripheral blood can be increased relative to a proportion of nucleated cells present in the sample of peripheral blood prior to enrichment forming a sample enriched in nucleated cells. The sample enriched in nucleated cells is then used for culture.
  • a proportion of nucleated cells is intended to refer to the percentage of nucleated cells, relative to the total number of cells, i.e. nucleated and non-nucleated cells, in the sample.
  • the language “increased relative to a proportion present ... prior to enrichment” is intended to mean that the percentage of nucleated cells present in the sample is greater after an enrichment procedure as compared to the percentage of nucleated cells present in the sample before the enrichment procedure.
  • the percentage of nucleated cells in a sample containing both nucleated and non-nucleated cells, such as peripheral blood can be increased relative to the starting sample by removing non-nucleated cells from the sample and/or by separating nucleated cells away from non-nucleated cells.
  • the language "a sample enriched in nucleated cells” is intended to include a sample derived from a maternal blood sample in which the percentage of nucleated cells present in the sample has been increased relative to the percentage present in the maternal blood sample prior to an enrichment procedure.
  • a sample enriched in nucleated cells is formed by separating non-nucleated cells from nucleated cells in a sample of peripheral blood obtained from a pregnant woman.
  • separating is intended to mean that non-nucleated cells and nucleated cells are isolated away from each other.
  • Non-nucleated cells can be separated from nucleated cells by density gradient centrifugation.
  • density gradient centrifugation is intended to refer to a technique in which a mixture of cells, e.g. cells of a blood sample, are centrifuged in a container, such as a centrifuge tube, through a material of a particular density to form layers containing different cell types.
  • density gradient centrifugation can be used to separate cells based upon differences in cell size and density.
  • Non-nucleated red blood cells have a different cell size and density than nucleated cells and thus can be separated from nucleated cells by density gradient centrifugation.
  • materials suitable for separating non- nucleated and nucleated cells by density gradient centrifugation are commercially available. These include Ficoll (Pharmacia, Uppsala, Sweden), Histopaque (Sigma Diagnostics, St.
  • centrifugation speeds and times have been determined by the manufacturers. Generally, appropriate centrifugation speeds and times are in the range of 400-500 g for 20-35 minutes at room temperature.
  • the maternal blood sample is typically separated into at least three layers: a supernatant layer containing serum and platelets; a mononuclear cell layer; and an agglutinated pellet which contains non-nucleated red blood cells.
  • a supernatant layer containing serum and platelets e.g. the mononuclear cell layer
  • an agglutinated pellet which contains non-nucleated red blood cells.
  • One or more cell layers containing nucleated cells and devoid of most non-nucleated red blood cells e.g. the mononuclear cell layer
  • the sample enriched in nucleated cells is then used for culture.
  • non-nucleated cells can be separated from nucleated cells by selective lysis of the non-nucleated cells.
  • selective lysis is intended to refer to preferential destruction of one cell type, e.g. non-nucleated cells, compared to other cell types, e.g. nucleated cells.
  • Cells in the maternal blood sample can be incubated in one of a number of hypotonic buffers known to be effective, and conventionally used, for lysing non-nucleated red blood cells, such as 0.17M NH4CI, 0.01M Tris, pH 7.3 (see e.g. Tiilikainen et al. Transplantation 17, 355 (1974) and Macera, et al. Leukemia Research 13, 729-734 (1989)). Buffers suitable for this purpose are also available commercially (e.g.
  • the resultant cell sample is a sample enriched in nucleated cells.
  • the maternal blood sample is manipulated before culturing cells in order to enrich for fetal cells, e.g., fetal erythroid progenitor cells, within the sample.
  • a proportion of fetal cells, e.g., fetal erythroid progenitor cells, present in the sample of peripheral blood can be increased relative to a proportion of fetal cells, e.g., fetal erythroid progenitor cells, present in the sample of peripheral blood prior to enrichment forming a sample enriched in fetal cells, e.g., fetal erythroid progenitor cells.
  • the sample enriched in fetal cells is then used for culture.
  • the language "a proportion of fetal cells, e.g., fetal erythroid progenitor cells” is intended to refer to the percentage of fetal cells, relative to the total number of cells, i.e.
  • nucleated and non-nucleated cells in the sample.
  • the language "increased relative to a proportion present ... prior to enrichment” is intended to mean that the percentage of fetal cells present in the sample is greater after an enrichment procedure as compared to the percentage of fetal cells present in the sample before the enrichment procedure.
  • the percentage of a particular type of fetal cells, e.g., fetal erythroid progenitor cells, in a sample containing other cell types, as in peripheral blood can be increased relative to the starting sample by removing other cell types, e.g.
  • a sample enriched in fetal cells is intended to include a sample derived from a maternal blood sample in which the percentage of fetal cells, e.g., fetal erythroid progenitor cells present in the sample has been increased relative to the percentage present in the maternal blood sample prior to an enrichment procedure.
  • a particular type of fetal cells e.g., erythroid progenitor cells
  • non-nucleated cells e.g. mature red blood cells
  • nucleated cells e.g. lymphocytes and mononuclear cells
  • density gradient centrifugation based upon differences in cell size and density of selected cell type and other cell types.
  • “dual density gradient centrifugation” is performed.
  • the language “dual density gradient centrifugation” is intended to include techniques in which a mixture of cells, e.g.
  • Dual density gradient centrifugation can be performed with the gradient materials described for single density gradient centrifugation (e.g. Ficoll, Histopaque, Nycodenz, Polymorphprep).
  • a sample enriched in fetal cells can be produced, for example, by density gradient centrifugation using Polymorphprep (Nycomed; Gibco BRL catalogue #100-1971).
  • Undiluted anticoagulated whole blood of a maternal blood sample can be centrifuged through the gradient material to form the following layers (from the top to the bottom of the tube): a supernatant layer containing serum and platelets; a mononuclear cell layer (the "buffy coat") containing lymphocytes and monocytes; a polymorphonuclear cell layer containing polymorphonuclear leukocytes, nucleated red blood cells and erythroid progenitor cells; and a non-nucleated red blood cell pellet.
  • the polymorphonuclear cell layer can be removed to form a sample enriched in fetal erythroid progenitor cells.
  • dual density gradient centrifugation using Histopaque can be used to produce a sample enriched in erythroid progenitor cells.
  • the nucleated red blood cell layer can be removed to form a sample enriched in erythroid progenitor cells.
  • Histopaque 1077 and 1119 can be used sequentially, as described in Example 1, to form a sample enriched in fetal cells, e.g., fetal erythroid progenitor cells.
  • the cell layers may not always be discrete and identifiable. Therefore, several different cell layers may be removed and ⁇ ultured separately in culture media containing a cell growth factor, e.g., an erythroid growth factor, e.g., erythropoietin, both to ensure that fetal cells, e.g.,erythroid progenitor cells have been properly selected and for comparison purposes (i.e., other cell layers can function as controls).
  • a cell growth factor e.g., an erythroid growth factor, e.g., erythropoietin
  • a sample enriched in fetal cells can also be produced using other techniques to remove non-nucleated cells and/or other cell types from a maternal blood sample or to enrich for erythroid progenitor cells.
  • antibodies specific for a cell surface marker present on a particular cell type can be used to either remove or enrich for that cell type within a maternal blood sample.
  • An antibody specific for a marker present on the selected fetal cell type, e.g., fetal erythroid progenitor cell can be used to select for erythroid progenitor cells, for example by flow cytometry, panning or immunomagnetic separation (positive selection of cells).
  • an antibody specific for a marker present on a cell type other than the selected fetal cell type can be used to remove that cell type from a maternal blood sample, for example by flow cytometry, panning or immunomagnetic separation (negative selection of cells) or by complement-mediated lysis of cells which have bound the antibody.
  • a sample enriched in nucleated cells or a sample enriched in fetal cells e.g., erythroid progenitor cells
  • a culture medium containing a cell growth factor e.g., an erythroid growth factor, e.g., erythropoietin
  • erythroid cells or cell colonies cannot be identified morphologically as being fetal-derived.
  • the method of the invention can further comprise detecting a fetal cell marker on or in the fetal cells.
  • fetal cell marker is intended to include a cell-surface, cytoplasmic or nuclear structure or molecule present on or in a fetal cell which can be used to distinguish a fetal cell from a maternal cell.
  • detecting a fetal cell marker is intended to include detection of the fetal cell marker itself or detection of another molecule which binds to the fetal cell marker.
  • a fetal cell marker can be detected on or in a fetal cell by, for example, contacting the cell, or portion thereof, with an antibody reactive against the fetal cell marker.
  • the antibody can be labelled with a detectable substance or can be further reacted with another molecule, e.g. a second antibody reactive against the first antibody, which is labelled with detectable substance.
  • Suitable detectable substances include enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
  • suitable enzymes include horseradish peroxidase, biotin, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase; examples of suitable enzyme/prosthetic group complexes include streptavidin and biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerytlirin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 1 ? I, 131 I, 35 S or J 3H. Standard immunohistochemistry techniques can be used to detect fetal cell marker expression using an antibody against the fetal cell marker.
  • Proteins which are expressed preferentially or exclusively on or in fetal cells as compared to maternal cells can be used as fetal cell markers and can be detected using an antibody against the protein.
  • suitable proteins which can be used as fetal cell markers include fetal hemoglobin and a fetal histone protein H°l .
  • Fetal hemoglobin is a cytoplasmic protein
  • fetal H°l is a nuclear protein.
  • an antigenic determinant which is preferentially or exclusively expressed on or in fetal erythroid cells can be used as a fetal cell marker.
  • An example of a suitable antigenic determinant which can be used as a fetal cell marker is the i antigenic determinant.
  • the i antigenic determinant is an unbranched membrane carbohydrate present on fetal erythroid cells.
  • Adult erythroid cells generally do not express i but rather express the I antigenic determinant, a branched carbohydrate.
  • Other examples of fetal cell markers include nucleic acid sequences which are unique to fetal cells compared to maternal cells. Examples of unique nucleic acid sequences include Y chromosomal DNA, in the case of fetal cells from a male fetus, and DNA encompassing a paternal polymorphism or polymorphic allele, such as an HLA antigen allele.
  • the method of the invention can further comprise detecting a nucleic acid sequence of interest in fetal nucleic acid of isolated fetal cells, e.g., fetal erythroid cells.
  • a nucleic acid sequence of interest is intended to include DNA, e.g. chromosomal DNA or a particular gene fragment within chromosomal DNA or amplified from chromosomal DNA, as well as RNA, e.g. mRNA or nuclear RNA.
  • fetal nucleic acid is intended to include fetal DNA and RNA, for example fetal chromosomal DNA and fetal mRNA.
  • Detection of a nucleic acid of interest in nucleic acid of fetal cells can be used to identify a fetal cell as being fetal-derived (i.e., the fetal nucleic acid can be a fetal cell marker) and also for analyses such as determination of fetal gender, detection of a genetic disease in the fetus and detection of a chromosomal abnormality in the fetus.
  • Fetal nucleic acid in fetal cells, cultured and isolated as described herein, can be analyzed for the occurrence of a nucleic acid of interest for diagnostic or other purposes.
  • Cultured fetal cells can be treated such that fetal nucleic acid present in the cells is made available for detection.
  • a nucleic acid sequence of interest can be detected in the available fetal nucleic acid by, for example, enzymatically amplifying the nucleic acid sequence of interest and/or by hybridizing a labelled nucleic acid probe to the nucleic acid sequence of interest.
  • the labelled probe used to detect a nucleic acid sequence of interest can be, for example, a labelled DNA probe, a labelled RNA probe or labelled oligonucleotides.
  • Fetal DNA can be made available by boiling the fetal cells to lyse them, thereby releasing fetal DNA, for instance prior to amplification of fetal DNA.
  • Fetal cells can be attached to a solid support, e.g. a microscope slide, in such a way that fetal nucleic acid is made available, for example by fixing fetal erythroid cells or erythroid cell nuclei to a microscope slide prior to in situ hybridization.
  • Fetal nucleic acid in fetal cells, or portion thereof e.g. nuclei
  • can be detected directly for example by in situ hybridization of a labelled nucleic acid probe complementary to a nucleic acid sequence of interest or the fetal nucleic acid can be amplified prior to detection using a known amplification technique such as the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Primers for PCR amplification are chosen which specifically amplify a DNA sequence of interest in the fetal DNA
  • a nucleic acid sequence which is to be detected in fetal nucleic acid of fetal erythroid cells is referred to herein as a "nucleic acid sequence of interest".
  • the nucleic acid of sequence of interest is a Y chromosomal DNA sequence.
  • the language "a Y chromosomal DNA sequence” is intended to include nucleotide sequences unique to the Y chromosome, including repetitive sequences. Detection of a Y chromosomal DNA sequence can be used both for detection of a fetal cell marker and for fetal gender identification.
  • the nucleic acid sequence of interest is a sequence of a gene associated with a disease-causing mutation.
  • a sequence of a gene associated with a disease-causing mutation is intended to include nucleotide sequences encompassing all or part of a gene which may contain a mutation which causes or is associated with a disease, or is linked to a gene which may contain a mutation which causes or is associated with a disease. That is, the detected gene sequence or a gene sequence linked to the detected gene sequence may contain a mutation which, if the mutation is present, causes or is associated with a disease. Detection of such a sequence can be used to determine if a fetus has the disease caused by or associated with the mutation.
  • the nucleic acid sequence of interest detects a chromosomal abnormality.
  • the language "detects a chromosomal abnormality" is intended to include nucleotide sequences which can detect changes in chromosome number, chromosomal deletions, chromosomal rearrangements and/or chromosomal alterations.
  • a nucleic acid sequence of interest which can detect a chromosomal abnormality can be, for example, a nucleic acid sequence specific for a particular chromosome, such as a repetitive sequence from a particular chromosome.
  • Preferred chromosomes to be examined for detection of a chromosomal abnormality include chromosomes 13, 18, 21, X and Y.
  • Chromosomal trisomies are most prevalent for these particular chromosomes.
  • Preparation of nucleic acid probes suitable for detecting chromosomal abnormalities can be made by standard techniques. For example see Klinger, K., et al. Am. J. Hum. Genet. 51, 55-65 (1992).
  • a nucleic acid of interest is detected in fetal nucleic acid of fetal cells by in situ hybridization.
  • In situ hybridization can be used both to detect chromosomal DNA and to detect cytoplasmic mRNA. See for example Lichter, P., et al. Hum. Genet. 80, 224-234 (1988) and U.S. Patent No. 4,888,278 to Singer et al. If in situ hybridization is to be carried out, fetal cells are separated onto a solid support, such as a microscope slide, such that fetal nucleic acid is available for detection.
  • In situ hybridization can be used, for example, to detect Y chromosome-specific sequences in fetal DNA in order to determine the gender of a fetus as described in greater detail in Example 3.
  • In situ hybridization can also be used to assess chromosomal abnormalities in a fetus, including chromosomal aneuploidies, such as a trisomy, or chromosomal rearrangements or deletions, as described in greater detail in Example 5.
  • fetal DNA is enzymatically amplifed prior to detection of a nucleic acid sequence of interest.
  • Fetal DNA can be amplified by PCR as described in detail in Example 4.
  • fetal erythroid cells can be lysed by boiling and fetal DNA can then be amplified for an appropriate number of cycles of denaturation and annealing (e.g., approximately 24-60).
  • Control samples include a tube without added DNA to monitor for false positive amplification.
  • more than one separate fetal gene can be amplified simultaneously.
  • This technique known as "multiplex" amplification, has been used with six sets of primers in the diagnosis of Duchenne's Muscular Dystrophy (Chamberlain, J.S., et al., Prenat. Diagnosis, 9, 349-355 (1989)).
  • the resulting amplification product may be a mixture which contains amplified fetal DNA of the sequence of interest (i.e., the DNA whose presence is to be detected and/or quantitated) and other DNA sequences.
  • the amplified fetal DNA sequence of interest and other DNA sequences can be separated, using known techniques, for example by gel electrophoresis.
  • Subsequent analysis of amplified DNA can be carried out using known techniques, such as: digestion with a restriction endonuclease, ultraviolet light visualization of ethidium bromide stained agarose gels, DNA sequencing, or hybridization with a labelled DNA probe, for example, specific oligonucleotide probes (Saiki, R.K., et al, Am. J. Hum. Genet., 43 (Suppl):A35 (1988)).
  • Amplification of and hybridization to allele-specific sequences can determine whether polymorphic differences exist between the amplified "maternal" and "fetal” samples and can be used to identify a female fetus based upon detection of paternal polymorphisms in the fetal DNA.
  • DNA sequences from the father can be identified in the autosomal chromosomes of the fetus. Consequently, the method of the present invention can be used to separate and identify female fetal cells, as well as male fetal cells, from maternal blood. Thus, the method can be used for all nucleic acid-based diagnostic procedures currently being achieved with other methods, such as amniocentesis.
  • the amplification mixture can be separated on the basis of the size of the nucleic acid fragments and the resulting size-separated fetal nucleic acid can be contacted with an appropriate selected nucleic acid probe or probes (nucleic acid sufficiently complementary to the nucleic acid sequence of interest that it hybridizes to the nucleic acid sequence of interest in fetal nucleic acid under the conditions used).
  • the nucleic acid probes are labelled (e.g., with a radioactive material, a fluorophore or other detectable material).
  • fetal nucleic acid/labelled nucleic acid probe complex can be detected and/or quantitated (e.g., by autoradiography, detection of the fluorescent label).
  • the quantity of labelled complex (and, thus, of fetal nucleic acid) can be determined by comparison with a standard curve (i.e., a predetermined relationship between the quantity of label detected and a given amount of nucleic acid present).
  • Fetal DNA/labelled probe complexes are subsequently detected, using a known technique, such as autoradiography.
  • Simple presence or absence of hybridization of the nucleic acid probe complementary to a nucleic acid of interest and the fetal DNA can be determined or the quantity of fetal DNA containing the nucleic acid sequence of interest can be determined.
  • the result is a qualitative or quantitative assessment of fetal DNA obtained from a maternal blood sample.
  • probes are available which detect a restriction-site polymorphism which is indicative of the presence of a disease-causing mutation within the gene.
  • Detection of such a restriction site polymorphism in fetal DNA using a nucleic acid probe specific for a gene associated with a disease of interest is indicative that the fetal DNA carries a mutation in the gene and therefore that the fetus has the disease.
  • the presence of fetal nucleic acid associated with diseases or conditions can be detected and/or quantitated by the present method.
  • an appropriate probe is used to detect the nucleic acid sequence of interest. For example, sequences from probes Stl4 (Oberle, I., et al., New Engl. J.
  • Stl4 is a highly polymorphic sequence isolated from the long arm of the X chromosome that can be used to distinguish female fetal DNA from maternal DNA.
  • the invention provides a method for isolating fetal erythroid cells in metaphase from a sample of peripheral blood from a pregnant woman.
  • the method involves obtaining a sample of peripheral blood from a pregnant woman, culturing cells within the sample of peripheral blood in a culture media containing an erythroid growth factor, e.g., erythropoietin, exposing cultured cells to an agent which inhibits progression of dividing cells through the cell cycle or synchronizes growth of cells and isolating fetal erythroid cells in metaphase.
  • agent which inhibit progression of dividing cells through the cell cycle are known in the art and include colcemid, colchicine and vinblastine salt.
  • Agents which synchronize growth of cells include bromodeoxyuridine, fluorodeoxyuridine and ethidium bromide. Following culture of cells, metaphase spreads can be prepared by standard methods and used for further analysis (see Example 2).
  • the invention provides a method for preferentially isolating fetal cells from a current pregnancy in a peripheral blood sample from a multiparous pregnant woman.
  • the method involves obtaining a sample of peripheral blood from a multiparous pregnant woman, culturing cells within the sample of peripheral blood in a culture medium containing an erythroid growth factor, e.g., erythropoietin, and isolating fetal erythroid cells to preferentially isolate fetal cells from a current pregnancy.
  • an erythroid growth factor e.g., erythropoietin
  • This method is based upon the short life span of erythroid progenitor cells (see Beck, W.S. Hematology, 5th edition, MIT Press: Cambridge, MA (1991)). Since the lifespan of erythroid progenitor cells is about three months, fetal erythroid cells cultured from a peripheral blood sample of a multiparous pregnant woman will be derived from erythroid progenitor cells of a current fetus rather than from a former fetus. This is an advantage of isolating fetal erythroid cells rather than another, longer lived, fetal cell type that may be present in a maternal blood sample.
  • lymphocytes may live as long as several years in peripheral blood (Schroder et al. Transplantation 17, 346 (1974); Ciaranfi et al. Schweizerische Medizinische Wegitz 107, 134-138 (1977)).
  • a fetal lymphocyte detected in a maternal blood sample of a multiparous pregnant woman could either be from the current fetus or from a former fetus, making gender identification and/or diagnosis of the current fetus problematic.
  • EXAMPLE 1 Culture of Erythroid Progenitor Cells from a Sample of Peripheral Blood from a Pregnant Woman
  • erythroid progenitor cells were cultured from samples of peripheral blood from pregnant women.
  • the peripheral blood samples were first subjected to density gradient centrifugations (single or dual) in order to eliminate most of the abundant non-nucleated red blood cells present in peripheral blood and to remove many of the maternally-derived nucleated cell types in the sample while also enriching for erythroid progenitor cells prior to the culturing step. This allowed for culturing of much fewer total cell numbers than were present in the original peripheral blood sample.
  • Maternal peripheral blood samples were obtained by venipuncture with informed consent from pregnant women planning to undergo pregnancy sonogram
  • the maternal blood sample was obtained before the amniocentesis was performed.
  • the maternal blood samples were received via overnight courier the day after they were obtained. After being inverted a few times to ensure mixing of the samples, 5 ml of undiluted blood was taken from each of three samples and layered atop a Polymorphprep gradient as explained below.
  • the remaining blood was diluted with an equal volume of phosphate buffered saline and 5 ml aliquots of the diluted blood were layered atop a Ficoll-Paque gradient, a Histopaque dual gradient, and a Nycodenz dual gradient.
  • a schematic diagram illustrating the typical relative distribution of erythroid progenitor cells and nucleated red blood cells along a density gradient is shown in Figure 1.
  • the buffy coat at the plasma/Polymorphprep interface from each of the blood samples was withdrawn with a Pasteur pipette and transferred to a clean tube. Then the polymorphonuclear cell layer (termed P2), which theoretically contained the nucleated red blood cells and erythroid progenitors, was transferred into another clean tube. An equal volume of sterile 0.45 % NaCl in distilled H2O was added to restore the cells to the proper osmolarity. The cells were diluted up to 12 ml in phosphate buffered saline, and cultured as described below.
  • Nycodenz was obtained in powdered form (Gibco/BRL), and 27.6 g of Nycodenz powder was reconstituted up to a total volume of 100 ml in buffered medium consisting of 5 mM Tris-HCl, pH 7.5, 3 mM KC1, and 0.3 mM EDTA in distilled H2O. This 27.6 % solution, at a density of 1.150 g/ml, was sterilized by autoclaving. Iso-osmotic NaCl diluent (density 1.003 g/ml) was made by mixing 0.75 g NaCl into 100 ml of the buffered medium described above.
  • the NaCl diluent solution was sterilized through a 0.22 ⁇ m filter.
  • the 27.6 % Nycodenz solution and NaCl diluent were mixed in the ratios of 3:2 and 1 :1 to yield two solutions having a density of 1.090 g/ml and 1.075 g/ml respectively.
  • a 2 ml aliquot of Nycodenz 1.090 g/ml in a 15 ml tube was carefully overlayered with 1.5 ml of Nycodenz 1.075 g/ml.
  • a 5 ml aliquot of blood diluted 1 : 1 in phosphate buffered saline was then layered atop Nycodenz 1.075 g/ml.
  • the tubes were spun for 30 minutes at 1500 x g in a tabletop centrifuge at room temperature. It was noted that this gradient combination resulted in cell bands which were much more diffuse than on other gradients.
  • the cell layers were contaminated with a large number of nonnucleated red blood cells.
  • a Pasteur pipette was used to harvest the cell layer at the interface of the plasma and the 1.075 g/ml gradient (termed Nl). Most of the 1.090 layer was harvested with a Pasteur pipette (termed N2); because there were so many cells present in this layer, not all of them were used. These cell fractions were placed into clean tubes.
  • the basic culture medium used consisted of 1.1 % methylcellulose (4000 centipoises; Sigma Chemical Co.), 30 % fetal calf serum (Sigma Chemical Co.), 0.1 mM 2-mercaptoethanol (Sigma Chemical Co.), and 1 % bovine serum albumin (BSA, Fraction V; Sigma Chemical Co.) in Iscove's Modified Dulbecco's Medium (IMDM; Sigma Chemical Co.). Cultures were supplemented with 2 units/ml of erythropoietin (Sigma Chemical Co.) to promote erythroid cell growth and maturation.
  • IMDM Iscove's Modified Dulbecco's Medium
  • methylcellulose was weighed into a one liter autoclavable bottle containing 190 ml sterile IMDM. The solution was mixed on a stirring plate while the methylcellulose went slowly into solution. The solution was autoclaved with the stir bar still in the bottle. After autoclaving, the methylcellulose formed a solid pink plug in the bottle. The bottle was shaken vigorously until the methylcellulose broke up into a slurry. As it cooled to room temperature, the methylcellulose went back into solution.
  • BSA at a final concentration of 10 % and pH of 7.6 was prepared by weighing out 10 g BSA and adding it slowly to about 60 ml distilled H2O in a small beaker on a stir plate. Meanwhile, 10 % NaHC ⁇ 3 was prepared by dissolving 10 g NaHCO3 in distilled H2O to a final volume of 100 ml in a volumetric flask and filter sterilizing through a 0.22 ⁇ m filter. The BSA solution was titrated to pH 7.6 with 10 % NaHCO3 while being continuously monitored with a pH meter. This solution was poured into a volumetric flask, brought up to 100 ml with distilled H2O, and filter sterilized first through a 0.45 ⁇ m filter and then through a 0.22 ⁇ m filter.
  • the desired cell fraction was rinsed twice in phosphate buffered saline, rinsed once in IMDM, and resuspended up to a total volume of 0.5 ml in IMDM.
  • This 0.5 ml of cell suspension was added to a 2.3 ml aliquot of the methylcellulose medium in a 15 ml conical centrifuge tube along with 0.2 ml of 30 units/ml erythropoietin, at a final concentration of 2 units/ml.
  • Tubes were vigorously vortexed to ensure complete mixing of contents, as well as to break up any cell clusters which could mimic colony formation.
  • the final product was of the consistency of thick syrup.
  • 1.5 ml of the medium containing cells was placed in each of two covered 35 mm Falcon plastic tissue culture dishes (Becton Dickson, Lincoln Park, NJ) within a 100 mm Falcon plastic dish (Becton Dickson).
  • a third, uncovered, 35 mm Falcon dish was placed within the 100 mm Falcon dish and partially filled with two ml of sterile distilled water to provide humidity.
  • the cover was placed on the 100 mm Falcon dish and the entire dish was transferred to a 37 °C incubator with a 5 % CO2 atmosphere.
  • CFU-E and BFU-E colonies are shown in Figures 2 and 3, respectively (the colonies depicted in the figures were isolated from fetal livers).
  • Table 1 summarizes the number of both BFU-E and CFU-E colonies obtained from culturing each cell fraction of a blood sample from a pregnant woman. The sample was fractionated using Ficoll-Paque (F), dual Histopaque (H), Polymorphprep (P), and dual Nycodenz (N) density gradients. Colonies from fraction N2 could not be seen or counted due to the presence of a large number of red blood eells which obscured the culture.
  • F Ficoll-Paque
  • H dual Histopaque
  • P Polymorphprep
  • N dual Nycodenz
  • erythroid cell colonies cultured as described in Example 1 were isolated and used to prepare metaphase spreads of erythroid cells on a microscope slide for further analysis.
  • the erythroid colonies were harvested directly onto Teflon-backed glass microscope slides (Cel-Line Assoc. Inc., Newfield, Nl) according to the basic method of Rajendra et al. (Human Genetics (1980) 55:363-366) and Dube et al. (Cancer Genetics and Cytogenetics (1981) 4:157-168). Because these erythroid cells are normally nonadherent, the slides were first coated with 0.1 % poly-L-lysine to induce the cells to attach to the slides so that they would not be lost during the harvesting procedure.
  • poly-L-lysine treatment 100 mg poly-L-lysine (Sigma Chemical Co.) was dissolved in 10 ml of distilled H2O to make a 1 % stock solution. Aliquots of one ml each were stored frozen until used.
  • 100 ⁇ l of 1 % stock solution was diluted with 900 ⁇ l of distilled H2O. A drop of the working solution was placed on each 5 mm site on each slide.
  • the slides were laid flat and nonoverlapping in a humid chamber (a sealed plastic box containing moist paper towels to provide humidity and a rack to keep the slides from contacting the paper towels) and incubated for two hours at 37 °C. After the incubation the slides were rinsed individually in a beaker of distilled water and air dried at room temperature. The coated slides were stored frozen at -20 °C until ready to use.
  • Colonies were identified under an inverted microscope and plucked from the medium with a Gilson Pipetman (Rainin Instrument Co., Inc., Woburn, MA) set to 4 ⁇ l, using a 10 ⁇ l tip. Each colony was dispersed by pipetting up and down 10 to 20 times into a 15 ⁇ l drop of IMDM containing 0.1 ⁇ g/ml colcemid (Gibco/BRL) on a poly-L-lysine coated slide. Slides were placed flat in a humid chamber at 37 °C for 15 minutes, then 20 ⁇ l of 0.075 M KC1 hypotonic solution was gently infused into each drop.
  • Gilson Pipetman Rosin Instrument Co., Inc., Woburn, MA
  • Erythroid cells on microscope slides prepared in this manner can be used for hybridization of the cells to a nucleic acid probe (e.g., fluorescent in situ hybridization), Wright's staining, banding or storage. Erythroid cells not arrested at the metaphase stage were prepared on microscope slides in the same manner by omitting colcemid from the cell- containing solution.
  • a nucleic acid probe e.g., fluorescent in situ hybridization
  • Wright's staining e.g., Wright's staining, banding or storage.
  • Erythroid cells not arrested at the metaphase stage were prepared on microscope slides in the same manner by omitting colcemid from the cell- containing solution.
  • EXAMPLE 3 Detection of Fetal DNA in Cultured Erythroid Cells by Fluorescent In Situ Hybridization
  • FISH flourescent in situ hybridization
  • Erythroid progenitor cells were cultured as described in Example 1 and colonies were isolated and placed onto microscope slides as described in Example 2.
  • FISH was performed using an X-chromosome specific probe derived from a cosmid containing a pericentromeric repeat sequence of the X chromosome and a Y specific probe derived from a cosmid pDP97 containing repetitive sequences. The probes are described in further detail in Klinger et al. Am. J. Hum. Genet. 51, 55-65 (1992).
  • the X-specific probe was labeled with biotin whereas the Y-specific probe was labeled with digoxigenin.
  • the biotinylated X chromosome probe was used at a concentration of 2.5 ng/ ⁇ l and the digoxygeninlabelled Y chromosome probe was used at a concentration of 5 ng/ ⁇ l.
  • the hybridization, washing, and detection protocols used were modified from Klinger et al. Am. J. Hum. Genet. 51, 55-65 (1992)and were as follows. Immediately before hybridization the slides were dehydrated through a 70%-80%-90%-100% ethanol series for one minute each and then air dried. Probe DNA for both the X and Y chromosomes was suspended in 50 % formamide/10 % dextran sulfate/6X SSC (standard sodium citrate) with 8 ⁇ g/ ⁇ l sonicated salmon sperm DNA (Sigma Chemical Co.) and 2 ⁇ g/ ⁇ l human Cot-1 DNA (Gibco BRL). A 2.5 ⁇ l drop of probe cocktail was added to each 5 mm site on the slides and covered with a glass coverslip.
  • the coverslips were not sealed to the slide surfaces.
  • the probe and the target nuclear DNA on glass slides were codenatured by placing the slides on a slidewarmer at 80 °C for five to six minutes. The slides were incubated in a humid box at 37 °C overnight. The following day the coverslips were removed and the slides were washed once in 50 % formamide/2X SSC pH 7.0 at 42 °C for ten minutes, and once in 0.1X SSC at 60 °C for ten minutes.
  • a total of 37 peripheral blood samples were obtained from consenting pregnant women between 10 5/7 weeks and 22 weeks of gestation.
  • Cells were fractionated from whole blood using either a dual Histopaque 1.077/1.119 g/ml density gradient (21 samples) or a Polymorphprep gradient (14 samples).
  • Two samples were fractionated on dual Histopaque, Polymorphprep, Ficoll-Paque and dual Nycodenz gradients.
  • Fractionated cells were cultured with erythropoietin as described in Example 1. In most cases, both the HI or PI and the H2 or P2 cell fractions were cultured.
  • XY colonies were identified by FISH in seven samples, yielding a male detection rate of 33.3%.
  • the number of XY colonies per sample ranged from one to eight (2.1 % to 33.3% of the total number of colonies analyzed for those cases).
  • From the 16 pregnant women who were actually carrying female fetuses as confirmed by cytogenetic analysis (not including sample 17, with a male and a female twin), no XY colonies were detected, yielding a false-positive rate of 0 %.
  • the gestational ages of the 37 pregnancies analyzed varied from 10 5/7 weeks to 22 weeks.
  • Sample 1 at 10 5/7 weeks, was the earliest pregnancy with a male fetus to be analyzed, and showed male cells in four of 120 colonies. The latest gestational age at which a male colony was cultured from a sample was 17 weeks (sample 18).
  • Table 3 relates the gestational age (GA) in weeks at which blood samples were obtained to the gradient fraction from which cultured colonies were harvested, the length of time (in days) that cells were cultured before harvesting, and the number and percent of the total number of colonies harvested which showed male cells by FISH.
  • the polymerase chain reaction was used to amplify Y-specific DNA sequences present in fetal erythroid progenitor cells cultured from maternal peripheral blood.
  • the polymerase chain reaction, or PCR which has a capacity for making 10" copies of rare target gene sequences, is used to amplify gene sequences in cultured erythroid progenitor cells.
  • PCR primers specific for repeated sequences present on the Y chromosome are used. Repeated sequences are selected because they create a stronger amplification signal from a rare male fetal cell.
  • Primers which hybridize to a region of the short arm of the Y chromosome, encompassed by probe Y411 (Given by Dr.
  • Y411 is identical to Y156 (Muller, U., et al., Nucleic Acids Res., 14, 1325-1329 (1986)), is repeated 10-60 fold, and is absolutely Y specific on Southern blots.
  • Two Y411 region-specific primers, primers 411-01 and 411-03, which are designed to amplify a 222 base pair (bp) sequence detectable with the Y chromosome-specific probe Y411, are used.
  • the primer sequences are described in detail in Bianchi, D.W., et al. Proc. Natl. Acad. Sci. USA 87, 3279-3283 (1990).
  • Other suitable primers are described in Wachtel, S., et al. Hum. Reprod. 6, 1466-1469 (1991).
  • DNA from male and female individuals is prepared in tenfold dilutions (1 pg to 1 mg) and amplified using the standard reagents, including reaction buffer, in the GeneAmpkit (Perkin-Elmer Cetus catalogue #N801-0055) on a Perkin-Elmer DNA Thermal Cycler. Measures are taken to prevent aerosol contamination of samples with male DNA. All PCRs are performed under sterile conditions, wearing gloves, and using positive displacement pipettes. All reagents are prepared in a sterile manner and incubated overnight prior to PCR with a restriction endonuclease having a digestion site within the target sequence.
  • the number of amplification cycles is varied between 18 and 30. Each amplification cycle consists of 1 minute at 94°C, 2 minutes at 60°C and 3 minutes at 72°C, with a 10 minute extension in the last cycle. Amplified DNA samples are electrophoresed on agarose gels, transferred to nylon filters, and hybridized to 32 P-labelled Y411 probe.
  • Erythroid progenitor cells are cultured and isolated as described in Example 1.
  • the cells Prior to amplification, the cells are lysed by boiling which makes the erythroid cell DNA available for amplification.
  • the erythroid cell DNA is amplified for the 222 bp sequence in probe Y411 as a demonstration that the cells are derived from the fetus in male pregnancies.
  • the conditions used, as described above, make it possible to detect a minimum of 100 pg of fetal DNA, or the equivalent of 15 fetal cells.
  • the limit of sensitivity can be improved by extending the number of cycles used in PCR.
  • PCR is carried out using primers derived from a single copy of sequence specific for the long arm of the Y chromosome, PY49a (Guerin, P., et al., Nucleic Acids Res. , 16, 7759 (1988)).
  • in situ hybridization using chromosome-specific probe sets is performed on fetal erythroid progenitor cells cultured from maternal blood in order to detect chromosomal abnormalities in fetal cells.
  • a DNA probe set specific for a particular chromosome that provides both good signal to noise ratios and good spatial resolution of the fluorescent signals is used in in situ hybridization.
  • Specific probe sets have been developed for five chromosomes frequently seen as liveborn aneuploidies, chromosomes 13, 18, 21, X and Y.
  • a probe for chromosome 1 is used as a control.
  • the chromosome 21 probe set is a three-cocmid contig containing 80 kb of nonoverlapping DNA.
  • the chromosome 18 probe set is a three-cosmid contig containing 109 kb of nonoverlapping DNA.
  • the chromosome 13 probe set is a three-cosmid contig containing approximately 97 kb of nonoverlapping DNA.
  • the X chromosome probe is a cosmid containing a pericentromeric repeat sequence.
  • the Y probe is pDP97, a repetitive clone (a 5.3 kb EcoRI Y fragment from cosmid Y97 subcloned into EcoRI site of pUC-13). All the probes are described in further detail in Klinger et al. Am. J. Hum. Genet. 51, 55-65 (1992).
  • erythroid progenitor cells are cultured from maternal blood as described in Example 1 and chromosomal abnormalities are assessed by performing in situ hybridization using chromosome-specific probes such as those described above. In situ hybridization is performed as described in Example 3 and in Klinger et al. Am. J. Hum. Genet. 51, 55-65 (1992) under suppression conditions (Cremer et al., Hum Genet. 80, 235-246 (1988); Lichter et al., Hum Genet. 80, 224-234 (1988)).
  • Hybridization of high copy number repeat sequences is suppressed by inclusion of total genomic human DNA, and the chromosomal specificity can be verified by hybridization to metaphase spreads.
  • Probes are labelled with biotin-UTP, hybridized under suppression conditions, and specific hybridization detected by conjugated streptoavidin-FITC, which shows as a single "dot" in the FITC image upon microscopic analysis.
  • conjugated streptoavidin-FITC which shows as a single "dot" in the FITC image upon microscopic analysis.
  • the individual probes can be differentially labelled to allow for them to be distinguished fluorescently.
  • one probe can be labelled with biotin-UTP and the another with digoxigenin-UTP.
  • the former probe can be detected with Cy-3 streptavidin while the latter can be detected with anti- digoxigenin-FITC.
  • the probes give sharp, punctate fluorescent signals in interphase cells that are easily discriminated and enumerated.
  • Diagnosis of a chromosomal abnormality is accomplished by comparing the hybridization of a chromosome-specific probe to DNA from fetal erythroid cells to hybridization of the same probe to DNA from normal cells (a normal control).
  • Normal cells which may be any cells which do not contain a chromosomal abnormality in the chromosome(s) being examined, can be hybridized at the same time as the fetal erythroid cells to provide a normal control.
  • maternal cells can be used as a normal control.
  • a previously established normal control can be used for comparison.
  • a common chromosomal abnormality a trisomy (in which three copies of a particular chromosome are present in a cell rather that the normal two), can be diagnosed by detection of three fluorescent signals for a particular chromosome, e.g. commonly chromosomes 21, 18 or 13, in a fetal erythroid cell as compared to only two fluorescent signals for the same chromosome in a normal control.
  • a sufficient number of hybridized cells are examined to make a statistically significant determination of the number of fluorescent signals present per cell.

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Abstract

L'invention concerne des procédés de culture et d'isolation de cellules f÷tales, par exemple de cellules f÷tales de la lignée érythrocytaire, à partir d'un échantillon de sang périphérique prélevé sur une femme enceinte. Ces cellules provenant de l'échantillon de sang périphérique sont mises en culture dans un milieu contenant un facteur de croissance cellulaire, par exemple un facteur de croissance érythrocytaire tel que l'érythropoïétine, en vue de la culture de cellules f÷tales. Avant la culture, les cellules anucléées peuvent être retirées de l'échantillon de sang périphérique et/ou les cellules f÷tales peuvent être enrichies. Des propagations de cellules au stade de la métaphase peuvent être préparées à partir de cellules f÷tales de culture. Un marqueur de cellule f÷tale ou une séquence d'acide nucléique présentant un intérêt peuvent être détectés dans les cellules f÷tales de culture. Ces dernières peuvent être utilisées pour déterminer le sexe du f÷tus et pour détecter des mutations pathogènes et des anomalies chromosomiques dans un f÷tus.
PCT/US1995/003906 1994-03-29 1995-03-29 Culture et isolation de cellules f×tales a partir de sang peripherique maternel WO1995026417A1 (fr)

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EP95916136A EP0804614A1 (fr) 1994-03-29 1995-03-29 Culture et isolation de cellules f tales a partir de sang peripherique maternel
JP7525275A JPH09510875A (ja) 1994-03-29 1995-03-29 母親の末梢血からの胎児細胞の培養および単離
AU22745/95A AU2274595A (en) 1994-03-29 1995-03-29 Culture and isolation of fetal cells from maternal peripheral blood

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Cited By (14)

* Cited by examiner, † Cited by third party
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WO1998002528A1 (fr) * 1996-07-12 1998-01-22 Domenico Valerio Isolement et culture de cellules foetales de sang maternel peripherique
WO2008048931A1 (fr) * 2006-10-16 2008-04-24 Celula Inc. Procédés et compositions pour le développement différentiel de cellules fœtales dans le sang maternel et leur utilisation
AU2005251379B2 (en) * 2004-06-01 2011-03-31 Kwalata Trading Limited In vitro techniques for use with stem cells
US8586306B2 (en) 2009-02-18 2013-11-19 Streck, Inc. Preservation of cell-free RNA in blood samples
WO2013192620A1 (fr) 2012-06-22 2013-12-27 Quantrx Biomedical Corporation Procédé d'obtention de cellules fœtales et composants cellulaires fœtaux
US20140220682A1 (en) * 2011-06-14 2014-08-07 The University Of North Carolina At Chapel Hill Isolation, Expansion and Use of Autologous Pluripotent Stem Cells
WO2016125924A1 (fr) * 2015-02-03 2016-08-11 한국기초과학지원연구원 Dispositif et procédé de refroidissement d'échantillon permettant une détection d'image liée par un microscope optique et un microscope électronique
US9956281B2 (en) 2011-05-04 2018-05-01 Streck, Inc. Inactivated virus compositions and methods of preparing such compositions
US10091984B2 (en) 2013-07-24 2018-10-09 Streck, Inc. Compositions and methods for stabilizing circulating tumor cells
US10966421B2 (en) 2002-10-16 2021-04-06 Streck, Inc. Method and device for collecting and preserving cells for analysis
US11168351B2 (en) 2015-03-05 2021-11-09 Streck, Inc. Stabilization of nucleic acids in urine
US11299764B2 (en) 2015-11-20 2022-04-12 Streck, Inc. Single spin process for blood plasma separation and plasma composition including preservative
US11506655B2 (en) 2016-07-29 2022-11-22 Streck, Inc. Suspension composition for hematology analysis control
US11634747B2 (en) 2009-01-21 2023-04-25 Streck Llc Preservation of fetal nucleic acids in maternal plasma

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CN101883851A (zh) * 2007-09-21 2010-11-10 生物概念股份有限公司 胎儿细胞和核酸的鉴定和分离
JP5527802B2 (ja) * 2009-11-24 2014-06-25 株式会社シームス 疾病予測システム
JP2016136921A (ja) * 2015-01-29 2016-08-04 大日本印刷株式会社 細胞培養容器

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WO1991016452A1 (fr) * 1990-04-23 1991-10-31 Cellpro Incorporated Procede servant a enrichir des cellules f×tales avec du sang maternel
WO1993008269A1 (fr) * 1991-10-23 1993-04-29 Cellpro, Incorporated Procede pour enrichir des cellules souches f×tales issues du sang maternel
US5275933A (en) * 1992-09-25 1994-01-04 The Board Of Trustees Of The Leland Stanford Junior University Triple gradient process for recovering nucleated fetal cells from maternal blood

Patent Citations (3)

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WO1991016452A1 (fr) * 1990-04-23 1991-10-31 Cellpro Incorporated Procede servant a enrichir des cellules f×tales avec du sang maternel
WO1993008269A1 (fr) * 1991-10-23 1993-04-29 Cellpro, Incorporated Procede pour enrichir des cellules souches f×tales issues du sang maternel
US5275933A (en) * 1992-09-25 1994-01-04 The Board Of Trustees Of The Leland Stanford Junior University Triple gradient process for recovering nucleated fetal cells from maternal blood

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998002528A1 (fr) * 1996-07-12 1998-01-22 Domenico Valerio Isolement et culture de cellules foetales de sang maternel peripherique
US10966421B2 (en) 2002-10-16 2021-04-06 Streck, Inc. Method and device for collecting and preserving cells for analysis
US11647743B2 (en) 2002-10-16 2023-05-16 Streck Llc Method and device for collecting and preserving cells for analysis
AU2005251379B2 (en) * 2004-06-01 2011-03-31 Kwalata Trading Limited In vitro techniques for use with stem cells
WO2008048931A1 (fr) * 2006-10-16 2008-04-24 Celula Inc. Procédés et compositions pour le développement différentiel de cellules fœtales dans le sang maternel et leur utilisation
US11634747B2 (en) 2009-01-21 2023-04-25 Streck Llc Preservation of fetal nucleic acids in maternal plasma
US8586306B2 (en) 2009-02-18 2013-11-19 Streck, Inc. Preservation of cell-free RNA in blood samples
US11761025B2 (en) 2009-02-18 2023-09-19 Streck Llc Preservation of cell-free nucleic acids
US9657227B2 (en) 2009-02-18 2017-05-23 Streck, Inc. Preservation of cell-free RNA in blood samples
US9926590B2 (en) 2009-02-18 2018-03-27 Streck, Inc. Devices and compositions for preservation of cell-free nucleic acids
US20180216165A1 (en) 2009-02-18 2018-08-02 Streck, Inc. Preservation of cell-free nucleic acids
US10294513B2 (en) 2009-02-18 2019-05-21 Streck, Inc. Preservation of cell-free nucleic acids
US10689686B2 (en) 2009-02-18 2020-06-23 Streck, Inc. Preservation of cell-free nucleic acids
US10144955B2 (en) 2009-02-18 2018-12-04 Streck, Inc. Methods for preservation of cell-free nucleic acids
US9956281B2 (en) 2011-05-04 2018-05-01 Streck, Inc. Inactivated virus compositions and methods of preparing such compositions
US9422526B2 (en) * 2011-06-14 2016-08-23 The University Of North Carolina At Chapel Hill Isolation, expansion and use of autologous pluripotent stem cells
US20140220682A1 (en) * 2011-06-14 2014-08-07 The University Of North Carolina At Chapel Hill Isolation, Expansion and Use of Autologous Pluripotent Stem Cells
US10792018B2 (en) 2012-06-22 2020-10-06 Preprogen Llc Method for obtaining fetal cells and fetal cellular components
US10058306B2 (en) 2012-06-22 2018-08-28 Preprogen, LLC Method for obtaining fetal cells and fetal cellular components
EP4008270A1 (fr) 2012-06-22 2022-06-08 Preprogen LLC Procédé d'obtention de cellules foetales et composants cellulaires foetaux
WO2013192620A1 (fr) 2012-06-22 2013-12-27 Quantrx Biomedical Corporation Procédé d'obtention de cellules fœtales et composants cellulaires fœtaux
US10674721B2 (en) 2013-07-24 2020-06-09 Streck, Inc. Compositions and methods for stabilizing circulating tumor cells
US10091984B2 (en) 2013-07-24 2018-10-09 Streck, Inc. Compositions and methods for stabilizing circulating tumor cells
US11547111B2 (en) 2013-07-24 2023-01-10 Streck, Inc. Compositions and methods for stabilizing circulating tumor cells
WO2016125924A1 (fr) * 2015-02-03 2016-08-11 한국기초과학지원연구원 Dispositif et procédé de refroidissement d'échantillon permettant une détection d'image liée par un microscope optique et un microscope électronique
US11168351B2 (en) 2015-03-05 2021-11-09 Streck, Inc. Stabilization of nucleic acids in urine
US11299764B2 (en) 2015-11-20 2022-04-12 Streck, Inc. Single spin process for blood plasma separation and plasma composition including preservative
US11506655B2 (en) 2016-07-29 2022-11-22 Streck, Inc. Suspension composition for hematology analysis control

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JPH09510875A (ja) 1997-11-04
AU2274595A (en) 1995-10-17
CA2183657A1 (fr) 1995-10-05
EP0804614A1 (fr) 1997-11-05

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