Cell, Vol. 5, 11-17, May 1975. Copyright01975 by MIT

D-Valine as a Selective Agent for Normal Human and Rodent Epithelial Cells in Culture

Scott F. Gilbert and Barbara Ft. Migeon Departments of Pediatrics and Biology Johns Hopkins University Baltimore, Maryland 21205

Summary

A nutrient medium has been developed to enable the growth of normal epithellal cells while selec- tively lnhlbltlng flbroblast proliferation. In this me- dium, D-vallne Is substituted for L-valine; and only those cells containing D-amlno acid oxldase can convert the D-amino acid Into Its essential L-enan- tlomer. The ablllty to select for cells with this en- zyme has enabled us to maintain eplthellal cell populatlons free from fibroblast overgrowth. The presence of D-amino acid oxidase has been hlsto- chemically conflrmed in the eplthellal cells select- ed from renal cell suspensions and explants. The ability to proliferate in the selective medlum Is transmitted to the clonal progeny of these cells. Moreover, epithelial cell proliferation In thls medl- urn indicates the presence of D-amlno acid oxl- dase, which we have detected In tissues where it had not previously been reported-fetal human kld- ney, lung, and cord. Flbroblasts will not grow In the selective medlum, but will proliferate normally If the product of the D-amino acid oxldase reactlon Is supplied.

Introduction

The application of cell culture to the analysis of mammalian development has been severely limited because of the proliferative advantage of fibroblasts over the epithelial cells of an organ. Observations that outgrowths from mammalian organ explants may be initially epithelial but are soon obscured by stromal fibroblasts (Carrel and Burrows, 1910; Fleischer and Loeb, 1911; Champy, 1913) led

Medium: D -va 1 he

Fischer (1946) to reflect that this was one of the major obstacles to the study of differentiated cells in culture. Studies of epithelial cells from explants can be performed only before fibroblastic out- growth (Pyke and Gelfand, 1974). We have formu- lated a medium which makes essential an enzyme not found in fibroblasts, thus enabling the selective proliferation of epithelial cells from various organs.

The selective medium is based on the require- ment of the essential amino acid L-valine for cell growth (Eagle, 1955; Litwin, 1974) and on the oc- currence of D-amino acid oxidase [D-amino acid: oxygen oxidoreductase (deaminating); EC 1.4.3.31 only in such specialized cells as the hepatocytes and renal tubular epithelium of many mammals, in- cluding man (Sallach and Fahien, 1969; Bliss, 1940). D-amino acid oxidase, which catalyzes the oxida- tive deamination of several D-amino acids, converts D-valine to 2-ketoisovaleric acid. This keto acid can then be converted to L-valine by the ubiquitous enzyme, branched-chain amino acid:2-oxoglu- tarate aminotransferase (Dancis et al., 1963; Ichi- hara and Koyoma, 1966; Shirai and Ichihara, 1971). Therefore as illustrated in Figure 1, cells with D-amino acid oxidase should be able to proliferate in nutrient medium where D-valine is substituted for L-valine (henceforth referred to as D-val; see Experimental Procedures), while fibroblasts, lack- ing a pathway for this transformation, should not survive. A control medium containing L-valine in- stead of D-valine, but otherwise identical to D-val, is referred to hereafter as L-val (see Experimental Procedures).

Results

Selection for Human Renal Eplthellal Cells Explants Fetal kidney explants in medium containing L-valine (either in the salt solution or in undialyzed fetal calf serum) produced outgrowths, initially

L-valine I I = I

Cell: w

D-Valine 2-Ketoisovaleric acid L-VZline

COOH FAD FADH COOH COOH I I I

//“\ NH

-> D-amino acid ’ =O <transaminase> c

oxida se I (CH3)2fi H 2 (CH3) 2 g ‘b H2N C(CH3)2

HH I Figure 1. Basis of Chemical Selection for Cells with D-Amino Acid Oxidase

Essential L-valine is removed from the nutrient medium and D-valine is substituted. Epithelial cells with D-amino acid oxidase convert D-valine to L-valine and therefore can proliferate while fibroblasts, lacking this enzyme, cannot.

Cell 12

Selection of Epithelial Cells by D. Valine 13

epithelial, which were subsequently obscured by the proliferation of fibroblasts. In D-val, however, fibroblast outgrowth was severely inhibited, and only epithelial populations proliferated from the ex- plants (Figure 2). In valine-free medium, some epithelial cells proliferated for the first two weeks; but subsequently there were many less cells in the valine-free medium than in D-val. The temporary growth in valine-free medium is most likely due to a residual intracellular L-valine pool. Single Cell Suspensions In growth medium, single cell suspensions from fetal kidney minces yielded clones at plating effi- ciencies from 0.30-0.95%. Although these were predominantly of the epithelial variety, fibroblast clones were also present. In regard to clonal mor- phology, each cell type “bred true.” Fibroblast clones always gave rise to progeny of fibroblastic morphology; and without exception, 160 subclones derived from 21 different epithelial clones main- tained their epithelial morphology.

In D-val, cells obtained from fetal human kidney minces yielded clones at an efficiency of approxi- mately 0.1%. These were entirely of the epithelial

type. When transferred to growth medium by clon- ing cylinders, these clones maintained their epithe- lioid appearance.

To determine if renal epithelial cells transmit the ability to grow in D-val, clones picked from D-val were maintained in growth medium until con- fluence. When these cells were replated into D-val, epithelial subclones were obtained. It is unlikely that proliferation in selective medium resulted from a large L-valine pool, as epithelial cells maintained in the selective medium for as long as four weeks produced clones when subsequently replated in D-val. Growth lnhibifion of Fibroblasfs That fibroblast growth in D-val was inhibited at cell densities as great as 105/60 mm dish was demon- strated for adult human skin fibroblasts as well as for cells from the established rodent cell lines, CHO, 3T3, Cl lD, and RAG. The effect of selection was more striking in rodent cells because the inhibited cells, unlike human fibroblasts, quickly detached from the plate. Furthermore, human cells can divide once or twice before they cease to proliferate. When the intermediate product, 2-ketoisovaleric

Figure 2. Human Fetal Renal Epithelial Cells Selected by D-Val (x 184)

(A and 8, opposite) Epithelial cell patterns which predominate during the first two to three weeks of culture. (C, above) Epithelial cells which predominate later. These patterns of cell growth are unique to cultured kidneys and persist in the absence of fibroblasts.

Cdl 14

acid (92 mg/l), was added to valine-free medium, both skin and renal fibroblasts were able to prolifer- ate as well as they did in L-val.

Enzyme Assay When assayed for FAD reduction using a D-amino acid as substrate, the kidney epithelial cytoplasm of D-val-selected cells displayed a strong purple color, whereas dermal fibroblasts could be seen in outline only. This is evidence that the pertinent en- zyme is the D-amino acid oxidase rather than a heretofore undetected racemase able to convert D-valine into L-valine directly.

Selection for Epithelial Cells from Other Sources The outgrowths from fetal lung and cord in growth medium, unlike those of the kidney, were predomi- nantly fibroblastic, with only rare epithelial cells. In D-vat, however, fibroblasts were inhibited shortly after their appearance; and by the end of the sec- ond week in culture, large areas of epithelial cells could be observed (Figure 4). This is surprising, as neither organ has been thought to contain signifi- cant amounts of D-amino acid oxidase.

Selection is not limited to human fetal tissues. D-val selected for epithelial cells, while L-val per- mitted the rapid proliferation of fibroblasts from adult mouse and rabbit kidney explants. In valine- free medium, proliferation from the explants was sparse.

Discussion

Several nonspecific chemical methods have been used to encourage the selective proliferation of epithelial cells in culture, including vitamin B com- plex (Heaton, 1926) hexenolactone (Medawar, Robinson, and Robinson, 1943), or steroids (Way- mouth, Chen, and Wood, 1971) as components of the nutrient medium. Media have also been devel- oped to favor the proliferation of cells with differen- tiated functions from tumor-derived HTC hepatoma (Kulka, Tomkins, and Crook, 1972) and murine neuroblastoma (Breakefield and Nirenberg, 1974). Normal hepatocytes have been selected from fetal rat liver by making essential the liver-specific en- zymes of the urea cycle (Leffert and Paul, 1972).

We have developed a nutrient medium specifical- ly favoring the growth in vitro of normal epithelial cells from various human and rodent organs on the basis of the cell’s ability to utilize D-amino acids. It is clear that D-val is not acting as a selective poison for fibroblasts because these cells prolifer- ate normally in medium containing D-valine as long as L-valine is included (Figure 3). This indicates that the absence of L-valine, rather than the pres- ence of D-valine, inhibits fibroblast proliferation.

Only epithelial cells proliferated in D-val, and this ability is transferred to the progeny of these cells. Furthermore, we have confirmed histochemically the presence of D-amino acid oxidase in the select- ed cells and have shown that fibroblasts supplied with the product of the D-amino acid oxidase reac- tion are able to proliferate. These findings suggest that the cells selected by D-val transmit to progeny not only their morphology, but also their ability to synthesize a “differentiated cell” enzyme in vitro.

This system enabled us to obtain epithelial clones and proliferation of epithelial cells not only from renal explants, but also from nonrenal tissues (that is, lung and cord) which rarely produce outgrowths of epithelial cells. Above all, these epithelial cell populations can be maintained free from fibroblast overgrowth.

D-val selection must be maintained to inhibit fi- broblasts in mixed populations of fibroblasts and epithelial cells or in dishes containing explants. Al- though many of the fibroblasts in D-val detach from the dish, the few which remain, even after weeks of L-val deprivation, are capable of rapid prolifer- ation when L-val is supplied. On the other hand, epithelial clones transferred as clones to dishes not containing fibroblasts may be maintained in nonse- lective medium.

Cell growth has been shown to be a sensitive indi- cator for the presence of an enzyme or substrate (Guthrie and Susi, 1963; Spector and Bloom, 1973). It is not surprising that by manometric methods, D- amino acid oxidase was not found in cultured mam- malian kidney cells (Burlington, 1959), since the rel- ative number of cultured cells containing this enzyme may be small. In vertebrates, D-amino acid oxidase was long thought to occur only in the kid- ney and liver (Krebs, 1935; Krebs, 1951) where ra- cemation of D-amino acids has been observed (Gibson et al., 1954). However, this enzyme has more recently been detected in human cerebellum (Neims, Zieverink, and Smilack, 1966) and neutro- phils (Cline and Lehrer, 1969). Our bioassay has enabled us to infer the presence of D-amino acid oxidase in the human fetal lung and cord-two places where it had not previously been found (Dunn and Perkoff, 1963)-and in the 10 week human embryonic kidney. This suggests that the enzyme may be more prevalent than is generally believed.

Investigations are currently underway to deter- mine if each of the epithelial cells favored by D-val is organ specific or if a common cell type (such as vascular endothelium) is being selected. Should the D-val-selected epithelial cells of renal origin have, in addition to a unique growth pattern, en- zymes found specifically in renal parenchymal cells, they will provide a model for studies of differentia-

Selection of Epithelial Cells by D-Valine 15

tion. Furthermore, the ability of renal epithelial cells to proliferate in a medium where established rodent cell lines cannot should permit the assignment of the gene for D-amino acid oxidase to a specific human chromosome.

Experimental Procedures

Specimens Kidneys and samples from other organs were obtained aseptically from -the dead products of therapeutic abortions of lo-20 weeks’ ges- tational age, -two 4 week-old Swiss albino mice. -and an adult New Zealand white rabbit. These specimens were placed in ice-cold sterile Hanks’ salt soiu- tion for less than an hour until cultures were initiated by cutting

Cl 1D

(IO31

RAG

organ minces into Lux plastic tissue culture dishes and flooding them with the appropriate medium. In the case of kidney samples, the capsule was removed prior to obtaining minces and cell sus- pensions. These ceil suspensions, where greater than 90% of the ceils were present as single cells, were prepared by the method of Leffert and Paul (1972) except that an equal volume of 0.25% trypsin (GIBCO) was added to the collagenase, and the cells were suspended in L-valine-free medium containing 15% dialyzed fetal calf serum.

Established rodent cell lines assayed for proliferation in D-val were Chinese hamster ovary cell line CHO K-l (CCLGl) and mouse renal adenocarcinoma RAG (CCL 142) from the American Type Culture Collection, and mouse lines 3T3 and Cl 1D contributed by M. Weiss.

Nutrient Media Growth medium was Eagle’s minimal essential medium (Earle’s salts) supplemented with nonessential amino acids and 15% fetal

I.-VALINE D,L-VALINE D-VALINE

Figure 3. Inhibition of Growth of Rodent Established Cell Lines by D-Val

Clones were obtained after 10 days from populations of 103 cells plated into L-val or into a 1 :l mixture of L-val and D-vat None were obtained in D-val. even from kidney-derived RAG cells.

Cell 16

Figure 4. Outgrowths from Lung Explants after 2 Weeks in L-Val or D-Val (x 230)

Proliferation of (A) fibroblasts in L-val, and (6) epithelial cells in D-val. Phase-contrast micrograph.

Selection of Epithelial Cells by D-Valine 17

calf serum (GIBCO). Medium selective for the growth of epithelial cells (D-val) was prepared by reformulating the growth medium salt solution, substituting D-val (92 mg/l) for L-val(46 mg/l). This solution was supplemented with (a) nonessential amino acids and (b) 15% fetal calf serum which had been dialyzed 20-28 hr at 4°C against three changes of 50 vol each of 0.85% NaCI. A third medi- um, L-val, was prepared in the same way as D-val (including the dialyzed fetal calf serum), but with L-valine (46 mg/l) instead of D-valine. In addition, a medium composed of D-valine salt solution and undialyzed fetal calf serum was prepared as a control for some studies.

Enzyme Assay Wohlrab’s method (1965) was used to stain for D-amino acid oxi- dase as modified for living cells in tissue culture by quick acetone fixation (Melnick, 1971). D-alanine was used as substrate since its reaction is catalyzed more efficiently than with D-valine (Krebs, 1951).

Cloning Clones were isolated with glass cloning cylinders (Ham and Puck, 1962).

Acknowledgment

This work was supported by a USPHS research grant. S.F.G. is a pre-doctoral fellow supported by an NIH training grant to the Department of Biology.

Received December 16, 1974; revised February 7, 1975

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