WO1994019487A1

WO1994019487A1 - Isolation of trophoblasts

Info

Publication number: WO1994019487A1
Application number: PCT/US1994/001555
Authority: WO
Inventors: Simon B. Goldbard; Larry A. Sklar; Babetta L. Marrone
Original assignee: Aprogenex, Inc.
Priority date: 1993-02-17
Filing date: 1994-02-10
Publication date: 1994-09-01
Also published as: IL108604A0; AU6174594A

Abstract

A method for isolating trophoblast cells in a sample of or derived from maternal blood by (i) optionally removing or lysing erythrocytes, such as by gradient separation from the sample; (ii) contacting the sample with a reagent that is reactive to produce a detectable response with an intracellular enzyme that is present in trophoblasts but not present in maternal blood cells; (iii) identifying trophoblasts by observing the detectable response; and (iv) isolating the trophoblasts from platelets and other cells in the sample. Where the enzyme is P450scc, the preferred reagent is 22-phenoxazonoxy-5-cholene-3β-ol and the response is fluorescence. Where the enzyme is 3β-HSD the reagent is preferably nitroblue tetrazolium with NAD+ in the presence of a 3β-hydroxy-Δ5-steroid substrate and the response is colorimetric.

Description

ISOIATION OF TROPHOBLASTS

Background of the Invention

The invention relates to a method for identifying and isolating trophoblasts in samples of or derived from maternal blood, thereby permitting safe and convenient examination of fetal cells for genetic defects.

An optical probe for the cytochrome P-450 cholesterol side chain cleavage enzyme is disclosed in Marrone, et al., U.S. Patent No. 5,110,725 and makes the observation of the conversion of cholesterol to pregnenolone, such aε by flow cytometry possible. Preliminary studies in granulosa cells have also shown that acute gonadotropin treatment increases resorufin fluorescence due to metabolism of the substrate, suggesting that the P-450 SC 7 probe can be used for the study of enzyme regulation. See also Marrone, et al., Endocrinology, 128:2654-2656 (1991)

The quantitative detection of conversion of pregnenolone to progesterone by 3B-hydroxysteroid dehydrogenase (3B-HSD) has been reported. Individual cell 3JJ-HSD was determined in digitonin permeabilized hen granulosa cells by dark blue staining observed under light microscopy in 3β-HSD active cells stained with NAD and nitroblue tetrazolium in the presence of exogenous enzyme substrate from the class of 3B-hydroxy- Δ -steroids, such aε pregnenolone or dehydroepiandroεterone. Marrone and Sebring in Biology of Reproduction, 40:1007-1011 (1989).

Fetal cells present in maternal circulation have both paternal and maternal alleleε and can be used for prenatal diagnosis of genetic diseases. The number of fetal cells (in this case trophoblasts) in material blood is very low (1 in 100,000) and the current state of the art does not allow the isolation of these cells for analysis-in a reliable way. Current approaches to fetal cell identification in maternal blood rely on the presence of cell surface epitopes (markers) that may be unique to fetal cells. Some of these surface epitopes are shared by maternal cells, complicating further the isolation of fetal cells. This subject was recently reviewed in Goldbard, Fetal Cells in the Maternal circulation: New Technology for Prenatal Diagnosis, Genetics & Teratology, vol. 1, no. 3 (1992).

The testing of prenatal abnormalities involves the use of invasive technologies (i.e., amniocentesis) that place the pregnancy at risk. A non-invasive procedure could allow all pregnant women to undergo testing for prenatal abnormalities.

SUMMARY OF THE INVENTION

The invention provideε a method used to isolate trophoblasts in samples derived from blood of women in their first or second trimester of pregnancy. Since thiε constitutes a non-invasive procedure with no danger to either mother or fetus, the test can be used for all pregnant women regardless of age and risk factors. Blood collection iε done at the doctor's office, clinic or elsewhere and cell isolation and analysis can be performed in similar facilities and in clinical genetics laboratories. The trophoblast isolation method of the invention iε used in conjunction with or, particularly, precedes and improves the samples provided for technologies like iri situ hybridization for the prenatal diagnosiε of genetic diseases.

This invention uses, instead of surface epitopes, several intracellular enzymes present only in the fetal trophoblastε in the maternal blood sample and not in any other cells of the sample. Thiε system eliminates several of the specificity problems encountered with the use of antibodies, simplifies and improves the identification and isolation of the desired trophoblast cells and provides a sample for subsequent testing from which other cell types have been removed.

Accordingly, the method provided by the invention is one for isolating trophoblast cells in a sample of maternal blood by contacting the sample with a reagent that iε reactive to produce a detectable response with an intracellular enzyme that is present only in trophoblasts and not in other cells of the sample; and isolating the trophoblasts εo detected from other formed elements (cells and platelets) in the sample. The isolated trophoblastε constitute an improved sample that can be analyzed for genetic disorders and the like, εuch as by in situ hybridization. Preferably the maternal blood sample is from peripheral maternal blood.

DETAILED DESCRIPTION

In one aspect, the invention relates to a method for isolating trophoblast cells in a sample derived from maternal blood by contacting the εample with a reagent that is reactive to produce a detectable response with an intracellular enzyme that is present in trophoblasts and not other cellε of the εample; iεolating the trophoblasts so detected from the εample. Preferably, the maternal blood is peripheral maternal blood.

In a preferred embodiment of this aεpect, the sample is essentially free of erythrocytes, such aε when the maternal blood εample iε pretreated by removal of the erythrocyteε by gradient separation. The gradient separation iε preferably performed on a polysaccharide pore gradient. The polysaccharide pore gradient is preferably a sucrose gradient. Examples of such gradients are the following: (a) Histopaque. Mixtures of polysaccharide and radiopaque contrast medium like Histopaque 1077 (SIGMA) which iε a solution of Ficoll (Pharmacia, Uppsala, Sweden) and sodium diatrizoate, adjusted to a density of 1.077g/ml. Most blood mononuclear cells will be retained at thiε density, facilitating their isolation from whole blood; (b) Percoll gradients. Percoll consists of colloidal silica particleε of 15-30nm diameter which have been coated with polyvinylpyrrolidone (PVP) . Percoll can form gradientε in the range of 1.0-1.3g/ml and can be also used effectively to isolate cells of different sizes from whole blood; (c) Dextran gradient. Mononuclear cells can also be separated from whole blood by using a 6% Dextran gradient. Such a sample can also be obtained by lyεing all nonnucleated cellε with an ammonium chloride solution prior to trophoblast detection. In another aspect, the invention provides for identifying as trophoblaεt cellε those cellε which are determined to have the intracellular enzyme P450 using a reagent having a cholesterol-based steroid connected through a linking group at the C-22 position with a chromophore effective to have a low optical response when attached to the steroid and a high optical response as an anion. The chromophore has a molecular size effective to not interfere with an enzymatic reaction that cleaves the chromophore from the steroid. The optical response is one of fluorescence.

In another aspect, the invention relates to detection of trophoblast cells by detecting the intracellular enzyme 3fi-hydroxysteroid dehydrogenaεe (3B-HSD). The 3B-HSD intracellular enzyme is detected in the presence of exogenous 3B-hydroxy Δ -steroid by a reagent comprising nitroblue tetrazolium and NAD .

In another aspect, the invention relates to a method for detecting trophoblaεt cells in a sample derived from maternal blood by contacting the εample to detect cells containing P450 with a reagent having a cholesterol-based steroid connected through a linking group at the C-22 position with a chromophore effective to have a low optical response when attached to the steroid and a high optical response as an anion, the chromophore having a molecular size effective to not interfere with an enzymatic reaction that cleaves the chromophore from the steroid; contacting the sample to detect 3B-HSD in the presence of exogenous 3B-hydroxy ___■ -steroid with a reagent comprising nitroblue tetrazolium and NAD . Preferably, the P450 iε detected fluorometrically and the 3B-HSD is detected colorimetrically. More particularly, it is preferred that 487 _.₇.

the P450 5CC iε fluorometrically detected by flow cytometry and 3B-HSD iε detected colorimetrically by light microscopy.

The reagent used in the present invention for detecting

P450 iε diεclosed in U.S. Patent No. 5,110,725. A s c cholesterol-like steroid is linked to a chromophore through a linking group to provide a compound of either Formula I or Formula II:

Formula I

Formula II

In order to bind to the enzyme, the steroid substrate has a 3(J-hydroxy group, unsaturation in the 5-6 bond, and has cholesterol-like stereochemistry at the C-17 and C-20α positions. The R position can be filled from the group consisting of H, alkyl,. and alkyl —CO—. As used herein, the term alkyl is limited to the low alkyl groups with 1-6 carbon atoms, i.e., methyl, ethyl, isopropyl, etc. The chromophore is selected to have advantageous spectral properties, i.e., to have little optical response when linked to the steroid substrate and an enhanced optical response when cleaved from the substrate and linking group. The chromophore molecule should also be of modest size to not interfere with the side chain cleavage reaction. A preferred chromophore iε selected from the xanthene dyes, linked as shown above, where R_ is H, halogen, alkyl, —0--alkyl, —COOalkyl, or —COOH; X is 0, S, NH, or N-alkyl; and Z is 0 or N,N-dialkyl.

Linking group L can have the structureε

wherein n=2 or 3 and X =0 or S.

The structural requirements of linking group L are not stringent, where the X' position can be filled from either 0 or S. Any one of the three linking groups may be selected, with a preferred link being the simple 0 or S link. The group containing an intermediate C provides a somewhat longer link and the group containing the (CH _,-_.) I provides longer linker arms. All three linking groups are expected to be cleaved with the P-450sec enzyme and to not interfere with the generation of the active chromophore.

Moεt preferred among these structures is 22-phenoxazonoxy-5-cholene-3D-ol, wherein R and R, are H, and L, X and Z are 0. The cholene-based steroid covalently conjugated at the C-22 position to resorufin and its analogs provides the P-450sec reagent. Aε a result of this conjugation the resorufin fluoreεcence is shifted and has a

40-fold lower quantum efficiency than the resorufin anion.

Specificity of the reagent for the P-450see enzyme is achieved by incorporating known structural and stereochemical features to the cholene-ring for enzyme recognition. Generation of a fluorescent signal is obtained by release of the resorufin moiety by the side chain cleavage enzyme.

Conventional flow cytometric analysis of activity of the P450 sec is performed in a two laser multiparameter flow cytometer in which the substrate fluorescence is green (maximum emission <-«• 560 nm) and the product fluorescence is red (maximum emission _*■_» 590 nm) (Marrone et al J. Org. Chem 56, 5393, 1991). Because the substrate can be excited with laser light over the range of 350 to 550 nm, and the product excited over the range of <~»• 475 to 575 nm, a single laser (514 or 528 nm) can be used to excite substrate and product simultaneously.

Isolation of the trophoblastε is preferably achieved by fluorescence activated cell sorting (FACS) using an automated cell sorting flow cytometer εuch as are available from Becton-Dickinson, Coulter and others.

-H-

EXAMPLE 1

Trophoblast Identification in Maternal Blood

A sample (20 ml) of peripheral blood from women in their firεt trimeεter is collected. The erythrocytes are lysed and the remaining cells are washed and resuspended in appropriate growth media. The cytochrome P-450 sec Probe is added to the cells at a concentration of luM, and the cells are incubated at 37 C for 60 minutes. Due to the fluorescence of the probe, individual trophoblaεts can now be identified and sorted out from other cells in the sample using a Vantage brand fluorescence activated cell εorter (Becton-Dickinεon and Company, Franklin Lakes, NJ) in which the substrate is excited with laser light over the range of 350 to 550 nm, and the product excited over the range of 475 to 575 nm. Thus, a single laser (514 or 528 nm) can be used to excite substrate and product simultaneously. Moreover, the origin of the sorted cells is further confirmed by treating the cells with a second substrate for the enzyme 3B-HSD. This is a colorimetric reaction performed on the sorted cells already deposited on slideε.

The positive cells for both enzymes are analyzed for the presence of the most frequent chromosomal aneuploidieε such as the ones from chromosomes X,Y,13,18, and 21. Analysis is performed by the use of fluorescent in-situ hybridization methodology. Briefly, labeled DNA probeε with specificity to the pertinent chromosomes, are hybridized to the cellε and detected with the uεe of fluoreεcent reagentε (i.e. avidin-FITC) . The number of copieε of each of these chromosomes can now be observed under the microscope, and the presence of aneuploidieε readily diagnoεed.

Claims

WHAT IS CLAIMED IS:

1. A method for isolating trophoblast cellε in a εample derived from maternal blood which compriεeε contacting the εample with a reagent that iε reactive to produce a detectable responεe with an intracellular enzyme that iε present in trophoblasts but not present in other cells of the sample; and iεolating trophoblaεtε producing the detectable response.

2. The method of claim 1 wherein the sample iε derived from peripheral maternal blood.

3. The method of claim 1 wherein, prior to contacting the sample with the reagent, any erythrocyteε therein are removed from the sample by gradient separation.

4. The method of claim 3 wherein the gradient separation is performed on a polysaccharide pore gradient.

5. The method of claim 4 wherein, prior to contacting the sample with the reagent, any erythrocytes therein are lysed.

6. The method of claim 1 wherein the intracellular enzyme is P450 and the sample is contacted with a reagent having a cholesterol-based steroid connected through a linking group at the C-22 position with a chromophore effective to have a low optical response when attached to the steroid and a high optical response as an anion, the chromophore having a molecular εize effective to not interfere with an enzymatic reaction that cleaves the chromophore from the steroid;

7. The method of claim 6 wherein the optical response is fluorescence.

8. The method of claim 6 wherein said linking group iε εelected from the group conεiεting of

where n = 2 or 3 and

X = 0 or S

9. The method of claim 6, wherein εaid chromophore is selected from the group consisting of

where R₇=H, halogen, alkyl,

—O-al yl, -COOalkyl, or —COOH X=0, S, NH, or N-alkyl Z=0 or N, N-dialkyl.

10. The method of claim 9 wherein said chromophore iε resorufin ether.

11. The method of claim 1 wherein the intracellular enzyme iε 3&-HSD and the reagent compriseε nitroblue tetrazolium and NAD in the preεence of a 3β-hydroxy

A -εteroid εubstrate.

12. A method for isolating trophoblast cells in a εample derived from maternal blood which compriseε contacting the εample with a reagent that iε reactive to produce a detectable response -with an intracellular enzyme that is present in trophoblasts but not present in other cells of the sample; identifying trophoblasts by observing the detectable response; and isolating the trophoblastε from the sample.

13. The method of claim 12 wherein the sample is derived from peripheral maternal blood.

14. The method of claim 12 wherein, prior to contacting the sample with the reagent, any erythrocytes therein are removed from the sample by gradient separation.

15. The method of claim 14 wherein the gradient separation is performed on a polysaccharide pore gradient.

16. The method of claim 15 wherein, prior to contacting the sample with the reagent, any erythrocyteε therein are lysed.

17. The method of claim 12 wherein the isolation is by automated fluorescence activated cell sorting.

18. A method for isolating trophoblast cellε in a sample derived from maternal blood which comprises:

(i) contacting the sample to detect P450 with sec a reagent having a cholesterol-based steroid connected through a linking group at the C-22 position with a chromophore effective to have a low optical response when attached to the steroid and a high optical response as an anion, the chromophore having a molecular size effective to not interfere with an enzymatic reaction that clearε the chromophore from the εteroid;

(ii) contacting the sample to detect 3J.-HSD with a reagent comprising nitroblue tetrazolium and NAD in the

5 presence of a 3B-hydroxy- -steroid substrate.

(iii) observing a detectable response to stepε (i) and (ii) ; and

(iv) isolating the trophoblasts from platelets and other cells in the sample.

19. The method of claim 18 wherein the P450sec is detected fluorometrically and the 3β-HSD is detected colorimetrically.

20. The method of claim 19 wherein the P450sec iε fluorometrically detected by flow cytometry, the 3P-HSD iε detected colorimetrically by light microscopy, and the trophoblastε so detected are isolated in a fluorescence activated cell sorter.