cultivated cells from mid-trimester amniotic fluids. iv. cell type identification via one and...

PRENATAL DIAGNOSIS, VOL. 2,79-88 (1982)

CULTIVATED CELLS FROM MID-TRIMESTER AMNIOTIC FLUIDS

IV. CELL TYPE IDENTIFICATION VIA ONE AND TWO- DIMENSIONAL ELECTROPHORESIS OF CLONAL WHOLE

CELL HOMOGENATES

PATRICIA JOHN ST ON*^$, DARRELL SALK*?, GEORGE M. MARTIN* AND HOLGER HOEHN*?

*Division of Genetic Pathology and Center for Inherited Diseases, University of Washington, Seattle, Washington, U.S.A.

TInstitut fur Humangenetik, Universitat Wurzburg, Federal Republic of Germany

SUMMARY

Clones of cultivated amniotic fluid cells that have distinct morphologic and growth characteristics (F, AF and E-type) were examined by one-dimensional SDS polyacrylamide gel electrophoresis (SDS-PAGE) and by two-dimensional electrophoresis employing isoelectric focusing and SDS-PAGE (IEF-PAGE). No qualitative differences in band pattern were observed in SDS-PAGE between the various amniotic fluid cell types, but consistent quantitative differences in the ratios of four bands of presumed filamentous proteins provided good distinction between amniotic fluid cells and postnatal skin fibroblast-like cells. By adding separation on the basis of electrical charge to that of molecular size (IEF-PAGE), we observed reproducible qualitative differences in the protein spot patterns between F and both AF and E-type amniotic fluid cells. At least eight discrete proteins appear not to be synthesized by prenatal F-type cells in comparison with their isogenic AF and E counterparts under identical culture conditions. The two-dimensional electrophoretic patterns thus confirm that F and AF amniotic cells, in spite of their morphologic and growth kinetic similarities, are developmentally distinct cell types that retain their differentiated states in culture.

KEY WORDS Prenatal diagnosis ; Amniotic fluid cell types ; Two-dimensional electrophoresis ; Polypeptide patterns

INTRODUCTION

We have previously proposed classifying clonable mid-trimester amniotic fluid cells into three major categories (F, A F and E) on the basis of morphological and growth kinetic criteria (Hoehn e f al., 1974, Figure 1). Any attempt at classification based solely on morphology and growth suffers from some degree of subjectivity reflecting specific conditions of cell culture that may prevail in different laboratories. We have therefore extended our analysis of the clonable fraction of amniotic fluid cells to a more objective biochemical level. As a first step in that direction we report the electrophoretic separation of whole cell homogenates of a series of independently isolated amniotic fluid cell clones. Postnatally derived skin fibroblast-like cell cultures serve as reference materials in some of these assays.

~ ~~

$ Addressee for correspondence: Holger Hoehn, M.D., Human Genetics, Koellikerstrasse 2, 8700 Wiirzburg, West Germany.

01 97-38 5 1 /82/020079-1 OSO 1 .OO 0 1982 by John Wiley & Sons, Ltd.

Received 6 June I981 Revised 18 October 1981

Accepted 9 November I981

80 P. JOHNSTON ET AL.

Figure 1. (a) Morphology of riving amniotic fluid cell cultures. F=fibroblast-like, AF= amniotic fluid, E=epitheloid type cells. Note superficial similarity between F and AF types at early stages of colony formation (5-8 days in culture; left panel). At 12-14 days of culture (confluent colonies) morphological differences between F and AF cultures become more apparent while those between AF and E-types

cultures decrease (right panel).

MATERIALS AND METHODS

F, AF and E-type amniotic fluid cell clones were isolated from primary amniotic fluid cultures using stainless steel cloning cylinders. Clonal isolates were grown in 35 mm plastic petri dishes and either collected directly for electrophoresis or expanded by subsequent transfers in 25 cm2 tissue culture flasks. Established cultures were stored in liquid nitrogen at densities of 1.5-2 x lo6 cells/ml of tissue culture medium in the presence of 10-16 per cent fetal calf serum and 8.6 per cent DMSO.

For one-dimensional analysis, electrophoresis employed the discontinuous SDS buffer system of Laemmli (1970) with some modifications. Confluent cultures in 35 mm dishes were harvested with a trypsin-versene solution (Martin, 1964), the suspended cells were washed twice in cold PBS in the presence of serum and once in cold PBS alone. Cell pellets were resuspended in 0.5 ml aliquots of Laemmli’s sample buffer, immersed in boiling water and cooled in an ice bath prior to loading on a poly-

AMNIOTIC FLUID CELL CLONES 81

Figure 1 (b) Morphology of h e d amniotic fluid cell cultures. Typical organization and growth patterns of F, A F and E cell types are most distinctive near clonal margins.

Figure 1 (c) Morphology of mature (14-16 days) amniotic fluid cell colonies. Typical discontinuous bull’s-eye pattern with narrow growth margins of AF-type colonies; central core and wide growth margins of E-type colonies; adult fibroblast-like ‘ribbing’ pattern without a defined central core

area of F-type colonies

acrylamide gel in a vertical slab gel apparatus (Hoefer Scientific 2001, San Francisco). The gel and electrode buffers contained 0.2 per cent SDS and the electrode buffer (pH 8.3) contained 0.05 M Tris and 0.384 glycine. The 17.5 x 30 cm x 1.5 mm contin- uous gradient gels were pumped from the bottom of the gel apparatus; the front mixing chamber of the gradient maker contained 30 ml of 6 per cent, and the rear chamber 60 ml of 15 per cent acrylamide solution. A water overlay was used to assure formation of a smooth separating gel surface 3.5 cm below the top of the apparatus. After polymerization overnight, gels were prerun for 5 h and a 3 per cent acrylamide stacking gel was cast using a teflon well-forming comb. Sample aliquots of 100 pl (con-


taining approximately 40 mg of total protein) were added to each well. Samples were electrophoresed for 16 h at 240V, 500pps (Ortec 4100 power supply; discharge capacitance 1.0 pF). After the run, gels were stained for 1.5 h in a water:methanol: acetic acid solution ( 5 : 5 : 1) containing 0.25 per cent Coomassie blue. The gels were destained overnight by washing in the same solution without Coomassie blue. Dried gels were scanned with a Beckman Quick Scan Densitometer at a wavelength of 520 lambda. Selected peak areas were quantitated with a Hewlett-Packard 9830A calcu- lator equipped with a map measuring device.

For two-dimensional electrophoresis, 5.0 x lo4 cells/cmZ were plated into Costar multiwell trays and the medium was changed during the second day after plating. Three days after plating, the cells were washed twice with methionine-free medium and then incubated for 2-3 h at 37°C in 200 p1 of fresh methionine-free medium containing 80 pCi of 3%-methionine (specific activity 850-1400 Cilmmole; Amersham-Searle). Whole cell extracts were prepared and processed as described by O’Farrel1 (1975) with slight modification. Approximately 1 x lo6 counts were loaded per gel. The isoelectric focusing gels were cast in pyrex tubes 14 cm long with an inside diameter of 4 mm; pH gradient determinations were made by aqueous dissolution and consecu- tive assays of 0.5 cm gel segments. A 12 per cent polyacrylamide separating gel, prepared the day before, was used for electrophoresis in the second dimension. Com- mercial molecular weight standards (200-25 Daltons ; Bio-rad, San Francisco) were co- electrophoresed in selected gels. Following electrophoresis the slab gels were fixed and dried (Hoefer Scientific Apparatus) and autoradiographs prepared with Kodak X-omat film (1-10 day exposures in the cold). The resulting protein spot patterns were compared by the ‘reference constellation’ method of Dewey et al. (1978): division of the gel area into distinct geographic domains with boundaries established by landmark proteins.

RESULTS

One-dimensional analysis (SDS-PAGE)

Figure 2 depicts a representative separation of whole cell homogenates on the basis of molecular size. Despite the visualization of 8 6 9 0 protein bands, there was no consistent qualitative difference among the band patterns of the respective prenatal cell types (F, AF, E). Examination of 57 gels also revealed no consistent qualitative differences between cultures of prenatal cells and skin fibroblast-like cells derived postnataily (PN) from other individuals. In contrast to this unexpected uniformity with respect to individual band position (molecular weight), we noted multiple differences in staining intensity of certain protein bands. Such quantitative differences occurred chiefly in the lower molecular weight range (less than 45 Daltons) and were most prominent among cultures with potentially large differences in the proportion of dividing versus resting cells (randomly studied cultures). With the exception of the four proteins discussed below, however, these quantitative differences were minimal if cultures were compared in a natuarlly synchronized resting state (confluency). The variations in staining intensity of four prominent bands in the intermediate and higher molecular weight ranges (designated 1-4 in Figure 2) appeared to occur independently of cellular growth. These variations were interdependent, a point that is substantiated by plotting the ratios of the densities of the two pairs of proteins (Figure 3). There is a surprisingly clear distinction between postnatal and prenatal cell homogenates with

AMNIOTIC FLUID CELL CLONES 83

Figure 2.One-dimensional electrophoresis in an SDS polyacrylamide slab gel, stained with Coomassie blue. Shown are whole cell homogenates of postnatal skin fibroblasts (PN) and amniotic fluid cell types E, AF, and F. Note the four landmark proteins with electrophoretic mobilities corresponding to (1) actin, (2) desmin and (3) myosin. The numbers at the left-hand margin indicate the respective positions of additional molecular weight markers, (94) phosphorylase, (86) bovine albumin, (53) glutamic dehydrogenase, (45) dog fish actin (a gift of Dr E. Fischer, Seattle), (39) tropomyosin, (14)

ribonuclea


( ? * . , , , , I

I0 I 5 Ratio of Protein #I to Protein #2

Figure 3. Quantitative relationships among the four landmark protein bands shown in Figure 2 as a function of cell type. The respective ratios represent comparisons of relative surface areas obtained with a map measuring device from densitometric gel tracings (see Materials and Methods). 0 : post-

natal skin fibroblasts (PN); 0 : F-type; 0 : AF-type; A: E-type amniotic fluid cells

respect to the combined ratios of these four landmark proteins. Furthermore, there is a suggestion of clustering among the prenatal cells: F-type cells are closest to the postnatal fibroblast-like cells and E-type cells are most distant. The spread of ratios among E-type cells is extensive, which is consistent with the considerable morphologic variation within the ‘epithelioid’ category of cultivated amniotic fluid cells. In part, the spread of ratio points might also reflect the non-stoichometric staining of proteins 1 and 2 that occasionally occurred due to excessive protein concentrations in some samples. This last point precludes a more concise analysis by means of gel densitometry .

Two-dimensional analysis (IEF- PA G E )

Figure 4 illustrates a typical ‘protein spot map’ of an AF-type amniotic fluid cell clone. A subset of invariable landmark spots has been circled and connected by lines. The spot patterns in the domains marked with bolder lines were examined in detail in 12 to 20 separations of each of the three cell types. Since we encountered technical variability in the quality of spot patterns from sample to sample and from run to run, it was necessary to evaluate multiple runs of multiple samples to determine consistent differences. A polypeptide spot was classified as ‘absent’ if it did not show up in repeated analysis and autoradiograph exposures longer than eight days failed to reveal its presence.

Figure 5 summarizes the pattern differences we used to differentiate among whole cell homogenates of cultivated F, AF and E-type amniotic fluid cells. In the ph 4-5 range, a characteristic chain of prominent polypeptide spots has been observed in all human cells tested so far (both pre- and postnatal). Among the amniotic fluid cells we studied, the AF cell type was found to regularly display a triplet formation in the middle portion of the left-hand ascending row, due to consistent differences in the intensity of spot number 2 (arrowed). Spot number 1, just below and to the left of spot number 2, was consistently less intense in E compared with AF-type cells; the spot marked 3 at the right-hand border of this area is apparently not synthesized in

ANMIOTIC FLUID CELL CLONES 85

prenatal F cells, but is made in varying amounts by both AF and E cells. The middle row of Figure 5 shows two areas selected from the pH 5-6 range: the presumptive actin cluster sits in the left-hand angle of the upper pentagon. There was a consistent absence of one prominent actin-associated protein in F-type cells (spot number 4, arrowed) and there were consistent intensity differences of three spots in AF and E- type cells (numbers 5-7). The spots l , 2, and 3 at the right upper margin of the pentagon were synthesized only by AF and E-type cells. In the adjacent hexagonal domain the spot labelled 8 and the unnumbered spot marked with an arrow are also absent, or greatly reduced, in F-type cells. The cluster on the right margin (three spots labelled 9) displays characteristic reproducible intensity differences between A F and E-type

Figure 4. Autoradiograph of two-dimensional gel electrophoresis of a whole cell homogenate of an 3%-methionine-labelled AF-type amniotic fluid cell colony. The polypeptide pattern is divided into regions defined by circled landmark spots. The four regions indicated with darker lines are shown in

detail in Figure 5

cells. Finally, spot number 1 in the pH 6-7 range is absent from F, but consistently present in AF.and E-type cells as part of a characteristic chain of proteins that also shows highly reproducible intensity differences between A F and E cell homogenates.

In summary, the detailed analysis of these four selected domains of the two- dimensional protein synthesis map reveals eight proteins that appear not to be synthesized by F-type amniotic fluid cells, or that are synthesized in only very small amounts below the level of autoradiographic detection (solid symbols in Figure 5). In contrast, AF and E-type samples yielded qualitatively very similar spot patterns. The latter two cell types can thus only be differentiated on the basis of quantitative intensity variations (open and dotted symbols in Figure 5). It should be emphasized that differences also exist in other regions of the gels and that in these illustrations we

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ANMIOTIC FLUID CELL CLONES 87

present only a selected group that was readily useful for differentiation. The possibility that the differences we observed result from genotype variation rather than from variation due to cell differentiation (Weil and Epstein, 1979) has been excluded by comparing sets of isogenic amniotic fluid cell clones isolated from a single amniotic fluid specimen. The possibility that the amniotic fluid F-type cells we studied repre- sented maternal cell contamination was excluded in at least two instances by demon- strating a male karyotype in the respective clonal isolate. Preliminary studies of synchronized and/or senescent postnatal fibroblast cultures suggest that the consistent differences observed in the protein maps of F versus A F and E-type cells are not due to the synthesis of different proteins associated with cell replication.

DISCUSSION

It is perhaps surprising that the striking morphological differences between fibroblast- like and epithelioid cell types do not seem to have qualitative biochemical correlates at the level of one-dimensional electrophoresis of whole cell extracts. The bulk of cellular proteins visualized with this method apperas to be shared by each of the cell types examined. One should not lose sight of the fact that the one-dimensional system provides a relatively crude level of biochemical phenotyping. One might expect to observe quantitative differences among proteins in differentiated cell types involving proteins that are known to play a major role in determining cellular morphology in vitro, e.g. the filamentous proteins. Indeed, based on comparison with molecular weight standards and published reports, the landmark bands 1, 2, and 3 that we analysed in one-dimensional gels probably represent actin, desmin, and myosin, respectively. We observed characteristic ratios among these proteins, which may reflect the basis of what we label morphologically as fibroblast-like or epithelioid cells. These findings could be explained either by differences in the relative amounts of the proteins present or by differences in solubility characteristics of the proteins in homogenates of the different cell types.

The two-dimensional analysis increases resolution by separating polypeptides on the basis of electrical charge in addition to molecular size. Furthermore, the method employs radioactive IabeIIing of newly-synthesized proteins rather than staining of the steady-state protein constitution. It is therefore likely that less abundant proteins contribute relatively more to the spot patterns in two-dimensional gels compared with the band pattern in one-dimensional gels, since the latter may be dominated by a small number of accumulated proteins. At this higher level of resolution we observed qualitative differences among as yet unidentified cellular proteins in F, AF, and E-type amniotic fluid cells. The most striking differences in the protein spot patterns were observed between the morphologically similar F and A F cell types. Even though the morphologic and growth kinetic distinctions between F and A F amniotic fluid cells are not as obvious as those between F and E cells, they are apparently developmentally distinct types that retain their differentiated states in culture. This concept has received ample support from studies performed in two laboratories: Priest et al. (1977, 1978, 1979) demonstrated biochemical, electron microscopic, and immunologic differences between cultured F and A F amniotic fluid cells; and Crouch and Bornstein (1978, 1979) and Crouch et al. (1978) have provided an elegant analysis of specific differences in procollagen and fibronectin secretion. These results stress the unique and distinctive nature of the so-called AF amniotic fluid cell type.


The question whether AF and E-type cells likewise represent ontogenetically distinct cell lineages remains unresolved. The E category of amniotic fluid cells is in itself morphologically heterogeneous (Hoehn et al., 1974), and it will thus be necessary necessary to examine various representative clones from this group to establish group specific markers. All one might say at the present time is that the great similarity in spot pattern between the AF and E-type amniotic fluid cells suggests a closer onto- genetic relationship between these cell types than that between F-type cells and either AF or E-type cells.

ACKNOWLEDGEMENTS

The authors are indebted to Clare Maxwell for her skilful isolation of amniotic fluid cell clones. This work was supported in part by NIH grants GM 15253, A G 01751 and DFG grant SFB 105.

REFERENCES

Crouch, E., Bornstein, P. (1978). Collagen synthesis by human amniotic fluid cells in culture: characterization of a procollagen with three identical proal (I) chains, Biochemistry, 17,

Crouch, E., Bornstein, P. (1979). Characterization of a type 1V procollagen synthesized by

Crouch E., Balian, G., Holbrook, K., Duskin, D., Bornstein, P. (1978). Amniotic fluid fibro-

Dewey, M.I., Filler, R., Mintz, B. (1978). Protein patterns of developmentally totipotent

Hoehn, H., Bryant, E.M., Karp, L.E., Martin, G.M. (1974). Cultivated cells from diagnostic

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human amniotic fluid cells in culture, J . Biol. Chem., 254, 4197-4204.

nectin. Characterization and synthesis by cells in culture, J. Cell Biol., 78, 710-715.

mouse teratocarcinoma cells and normal early embryo cells, Dev. Biol., 65, 171-182.

amniocentesis in second trimester pregnancies. I. Clonal morphology and growth potential, Pediatr. Res., 8, 746-754.

Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of head of bacteriophage T 4, Nature, 227, 680-685.

Martin, G.M. (1964). Use of tris (hydroxymethyl) aminomethane buffers in cultures of diploid human fibroblasts, Proc. SOC. Exp. Biol. Med., 116, 167-171.

O’Farrell, P.H. (1975). High resolution two-dimensional electrophoresis of proteins, J . Biol. Chem., 250,4007-4021.

Priest, R.E., Priest, J.H., Moinuddin, J.F., Keyser, A.J. (1977). Differentiation in human amniotic fluid cell cultures I: collagen production, J. Med. Genet., 14, 157-162.

Priest, R.E., Masimuthu, K.M., Priest, J.H. (1978). Differentiation in human amniotic fluid cell cultures: ultrastructural features, Lab. Invest., 39, 106-109.

Priest, R.E., Priest, J.H., Moinuddin, J.F., Sgontas, D.S. (1979). Differentiation in human amniotic fluid cell cultures: chorionic gonadotropin production, In Vitro, 15, 142-147.

Weil, J., Epstein, C.I. (1979). The effect of trisomy 21 on the patterns of polypeptide synthesis in human fibroblasts, Am. J. Hum. Genet., 31, 478-488.

cultivated cells from mid-trimester amniotic fluids. iv. cell type identification via one and...

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