Proc. Natl. Acad. Sci. USAVol. 78, No. 6, pp. 3659-3663, June 1981Cell Biology

Differentiation of fetal liver cells in vitro(hepatocyte/hydrocortisone/a-fetoprotein)

AARON E. FREEMAN*, EVA ENGVALLt, KOICHI HiRA*, YUTAKA YOSHIDAt, RANDALL H. KOTTELt,VIRGINIA HILBORN*, AND ERKKI RUOSLAHTIttLU Jolla Cancer Research Foundation, 2945 Science Park Road, La Jolla, California 92037; and *Center for Neurologic Study, 11211 Sorrento Valley Road, Suite H,San Diego, California 92121

Communicated by Robert J. Huebner, March 3, 1981

ABSTRACT Fetal mouse liver hepatocytes proliferate on asubstrate ofirradiated pigskin epidermis scored with scalpel bladeslits to permit cell access to the basement membrane. At the timethe cells are explanted, fetal genes, such as those responsible forproduction of a-fetoprotein (AFP) and y-glutamyltransferase(GGTase), are strongly expressed. The levels ofGGTase decreaserapidly and become undetectable within 2 weeks. The levels ofAFP decrease more gradually but become undetectable after 3-5weeks in culture. As the AFP levels decrease, there is a concom-itant increase in albumin production. Hydrocortisone prolongsproduction of AFP (for up to 8 weeks) but not of GGTase, and itdecreases albumin production for up to 8 weeks. Once cells loseAFP expression, addition of hydrocortisone does not restart it.Based on these data, fetal mouse liver hepatocytes, cultured onpigskin, seem to be an excellent in vitro model for liver cellmaturation.

It is well established that fetal cells often produce a set of geneproducts which, as the cells mature after birth, is replaced bya different set of gene products. Normally, the fetal gene prod-uct is never expressed again in quantity. When the fetal genedoes become expressed in the adult, it may represent diseaseor oncogenesis. The liver is of particular importance for theunderstanding of the relationship between differentiation andcarcinogenesis. Liver carcinogenesis has been extensively stud-ied, and a-fetoprotein (AFP) and albumin are important de-velopmental markers in the system. y-glutamyltransferase(GGTase) is another fetal marker in the liver. In the mouse,GGTase is expressed in the fetus and in the liver ofthe newbornanimal but usually disappears within 3 days after birth (1, 2).AFP is structurally related to albumin but the two proteins

are immunologically distinct (3, 4). The production rates ofAFPand albumin in the fetal liver change reciprocally so that AFPdecreases and albumin increases as development progresses(5). The predominant sources of AFP in the fetus are first theyolk sac and later the fetal hepatocytes (6). Levels of AFP upto 1-3 mg/ml in human fetal serum can be found at 12-20 weeksof gestation. These levels decrease as the fetus ages; after birth,AFP production in the normal liver virtually ceases. Disap-pearance of AFP is associated with a concomitant increase inalbumin production. Similar patterns ofAFP and albumin syn-thesis are seen in the mouse. Generally, AFP is a product offetal hepatocytes and albumin a product ofmature hepatocytes,but both proteins can be made by the same cells at the sametime (7).

When AFP is produced in the adult, relatively low levels areoften indicative of hepatocyte regeneration due to disease pro-cess. High levels may indicate hepatocarcinogenesis (for re-views ofAFP, see refs. 8 and 9).We wanted an in vitro model of hepatocyte maturation to

facilitate studies on the use of oncodevelopmental gene prod-ucts as early markers ofcell transformation and in studies relatedto developmental biology. Our survey of the literature did notreveal a satisfactory system: the period ofviable primary culturewas too short (10-12) or the cell lines derived were represent-ative of already differentiated adult hepatocytes (13-15) or ofhepatocarcinoma cells (16-18).We have developed a method for culturing primary mouse

liver cells for 10-12 weeks on a pigskin epidermal substrate (2,19). We find that fetal liver cells grown under these conditionsmaturate into an adult phenotype, much as liver cells in vivo,as evidenced by loss of AFP production and appearance of al-bumin production in the cultures. Maturation can be modulatedby addition of hydrocortisone to the culture medium. This invitro model may be useful in studies on control of differentiation.

MATERIALS AND METHODSCell Culture. Cells were cultured on a pigskin substrate as

described (2, 19). Briefly, the livers offull-term fetal NIH Swissmice (Microbiological Associates, Bethesda, MD) were mincedwith scissors into small bits approximately 1 mm in diameter.Twenty liver bits were placed on the epidermal side of tissueculture-quality sterile pigskin (Bum Treatment Skin Bank,Phoenix, AZ) previously prepared as follows. The pigskin wasthawed and then soaked for at least 14 days in Eagle's minimalessential medium containing 10% fetal bovine serum (desig-nated herein "E medium/serum"). After soaking, the 10-cmsquares of pigskin were trimmed to fit onto stainless steel gridswithin 90-mm organ culture dishes. With a sharp scalpel, manycriss-crossing slits were cut into the pigskin epidermis. Theneach pigskin square was placed on the grids epidermal side up.Culture medium was placed into the Petri dish so that the fluidlevel touched but did not overflow the pigskin substrate. Theculture medium was E medium/serum further supplementedwith 5-fold concentrated essential and nonessential amino acids.Cultures were incubated in 5% C02/95% air at 37°C. The cellswere fed three times a week. Supernatants were collected forassay of various proteins at 3 days after feeding.

Morphological Examination. The pigskin specimens andcells growing on them were cut into sections (2-4 mm) with

Abbreviations: AFP, a-fetoprotein; GGTase, y-glutamyltransferase.

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3659

3660 Cell Biology: Freeman et al.

blades on a paraffin board and put into either 10% formalin orcold acetone. The specimens put into acetone were stirred for3 hr with three changes of the solution. Fixed tissue then wassoaked in benzene for 45 min with agitation, put into benzene/paraffin for 15 min, and embedded in low-melting-point(-520C) paraffin. Serial 5-gm sections of paraffin blocks wereused to relate detailed structure and the localization of AFP,albumin, and enzyme activities.

Histochemical and Immunohistological Studies. Tissuespecimens fixed with cold acetone were used for enzyme stain-ing and immunohistological studies. Enzymatic staining forGGTase (20) and immunoperoxidase staining with peroxidaseand antiperoxidase complexes (21) were performed by pub-lished methods.

Rabbit antiserum to mouse albumin was purchased fromCappel Laboratories (Cochranville, PA). This antiserum gavea single line of precipitation against normal mouse serum. Rab-bit antiserum to mouse AFP was produced in this laboratory(22). This antiserum gave a single line of precipitation againstAFP-containing extracts of mouse embryos and purified AFPin immunodiffusion. It did not react with normal mouse serum.Normal rabbit preimmune serum was used as control.GGTase Assays Individual explants were removed from the

pigskin substrate with tweezers and suspended in 1.0 ml of0.05M Tris HCl buffer at pH 8.4. The suspension was sonicated for30 sec with a Bronwill sonicator equipped with a narrow-tipprobe. Activity ofGGT was determined by the method of Szasz(23) with a Sigma assay kit (Sigma, technical bulletin no. 416).Briefly, this method measures the GGTase-catalyzed reactionbetween y-glutamyl-p-nitroanilide as a substrate and glycylgly-cine as an acceptor to form glutamylglycylglycine and p-nitro-anilide. The rate of p-nitroanilide formation measured at 405nm is proportional to GGTase activity. Protein content in thesamples was determined by the method of Lowry et al. (24).

Quantitation of AFP and Albumin. AFP and albumin weremeasured by sandwich enzyme immunoassays (25). Mouse AFPused in these assays was purified from extracts ofmouse fetusesby immunoadsorbent chromatography (22).

Mouse albumin was purchased from Cappel Laboratories.Purified goat antibodies to AFP and an IgG fraction preparedfrom rabbit anti-albumin serum (Cappel Laboratories) by affin-ity chromatography on protein A-Sepharose (Pharmacia) wereused as antibodies in the assays, respectively.

RESULTS

Evidence for Maturation of Fetal Hepatocytes in Vitro. Theoriginal fetal liver explants could survive on pigskin epidermisfor several weeks. Cells migrated or grew from the explants intothe scalpel blade slits and undermined the pigskin epidermiswhich then exfoliated. The resulting cultures were 5-20 cellsdeep and consisted mostly of cells with round nuclei and largecytoplasm rich in organelles. Usually the cells did not aggregateinto any recognizable pattern; however, on rare occasion, duct-like structures lined with columnar epithelium could be ob-served. These cell sheets continued to survive and proliferatefor 12 weeks.

The cells were initially positive for GGTase by both bio-chemical and histochemical analyses but became negativewithin 1-2 weeks in culture (Fig. 1). High levels ofAFP werefound in the medium for 2-3 weeks; then AFP production de-clined rapidly and became undetectable by 5 weeks after ini-tiation of the cultures (Fig. 2). Albumin production was unde-tectable at first by immunoassay of culture medium, becamedetectable at the 10 ng/ml level after 3 weeks, and then in-creased rapidly to reach a maximum of about 1000 ng/ml after

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FIG. 1. GGTase staining in fetal liver and liver cultures. PD, pig-skin dermis; PE, pigskin epidermis; L, liver cells. (a) Original fetalliver, showing strongly positive activity in a pericanalicular pattern.(x375.) (b) Explant at 1 week showing weakly positive staining in apericanalicular pattern. (x252.) (c) Culture at 2 weeks, showingmostly negative staining. (X252.)

6 weeks. By immunohistochemical assay, however, albumincould be detected from the initiation of culture. Repeated ex-periments yielded similar results.

Modulation of Hepatocyte Maturation by Hydrocortisone.When hydrocortisone at 4 jg/ml was incorporated into the

Proc. Natl. Acad. Sci. USA 78 (1981)

Cell Biology: Freeman et al. Proc. Natl. Acad.. Sci. USA 78 (1981) 3661

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FIG. 2. Effect of hydrocortisone on AFP (a, *) and albumin (A,A) synthesis as fetal liver cells age in culture. AFP and albumin in themedium were quantitated (ng/ml) by sandwich enzyme immunoassayat various times of culture (weeks). Open symbols, data from regularcultures; solid symbols, data from cultures grown in presence ofhydrocortisone.

medium, the cells produced high levels ofAFP into the mediumfor 8-12 weeks (Fig. 2). Removal ofhydrocortisone after 3 weeksresulted in a rapid loss ofAFP production. Addition of hydro-cortisone to the medium did not restore AFP production in cul-tures previously grown in the absence of hydrocortisone for 3weeks.

In order to determine how AFP levels in the supernatantwere related to AFP levels in the cells themselves, we removedthe supernatants, solubilized whole cultures with Tween 80,and measured AFP in the solution. Six-week-old cultures grownon hydrocortisone had, on average, about 1000 ng of AFP perculture; replicate cultures grown in the absence of hydrocor-tisone had about 50 ng ofAFP per culture.

With hydrocortisone in the medium, albumin synthesis wasnot prevented but was decreased by 50% or more throughoutthe course of the experiments. Addition of hydrocortisone tocultures previously grown in its absence reduced albumin pro-duction. Cultures grown in hydrocortisone from day 0 and cul-tures grown in hydrocortisone from day 21 both producedequivalent amounts of albumin after 6 weeks. Hydrocortisonedid not prolong the expression of GGTase in the cultures be-yond what was seen without it.

After 6 weeks, all cultures, whether grown on hydrocortisoneor not, incorporated similar numbers of counts from[3H]thymidine into DNA (data not shown).

Shifting Cell Functions. Histochemical analyses of the cellsfor AFP and albumin revealed cells that stained for both AFPand albumin, some cells that stained for AFP only, and othersthat stained for albumin only. In 1-week-old cultures withouthydrocortisone, 50-80% ofthe cells contained AFP. Many cellswere positive for albumin also, but only a few cells were stronglystained. In 5-week-old cultures, in the absence of hydrocorti-sone, few cells contained AFP (Fig. 3a) but in the presence ofhydrocortisone, 50-80% of the cells contained AFP (Fig. 3b).In 5-week-old cultures, 50-80% of the cells were stronglystained for albumin whether or not hydrocortisone was presentin the medium (Fig. 3c).

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livercultured with hydrocortisone In the presence of hydrocortisone,*1, - --41 -; i

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FIG. 3. Immunohistochemical staining of AFP and albumin in 5-week-old liver cultures. (a) AFP in liver cultured without hydrocor-tisone. Note that there is little positive staining. (X252.) (b) AFP inliver cultured with hydrocortisone. In the presence of hydrocortisone,AFP activity is retained. (x 535.) (c) Albumin in liver cultured withouthydrocortisone. This micrograph shows a section from the same cultureas in a. Note that the albumin-positive cells are growing between thepigskin dermis and epidermis. (x535.)

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3662 Cell Biology: Freeman et al.

Fetal lung tissue cultures on pigskin did not show any stainingwith either anti-albumin or anti-AFP. When normal rabbitserum was substituted for the antisera, no staining was obtainedwith any of the cultured tissues.

DISCUSSION

Our results show that fetal mouse liver cells growing on pigskinepidermis maturate in a manner similar to fetal hepatocytesafter birth. As the cells aged in culture, the amount of the fetalproteins AFP and GGTase decreased and the amount of albu-min increased.

The system we describe is simple and consistently repro-

ducible. Virtually 90% of fetal mouse liver explants in everyexperiment formed a viable proliferating cell colony. The grad-ual loss of AFP expression in our cultures is not due to a re-

placement of liver cells by other cell types because there was

a reciprocal increase in the amount of albumin in the culturemedium. Moreover, after 5 weeks in culture, albumin could bedetected in 50-80% of the cells, indicating that hepatocytescontinued to be the major cell population in these cultures. Thehepatocytes in our cultures without hydrocortisone did not pro-

duce detectable amounts of AFP after 4-5 weeks in culture.These results are consistent with a previous report by Parsa andFlancbaum (26) that embryonic rat liver rudiments grow on

Millipore filters and undergo a normal ontogenesis of plasmaproteins. That elegant report was based on electrophoretic tech-niques and did not present quantitative data on growth or geneproduct expression; however, it seems clear that the maturationof fetal liver cells in vitro is a natural phenomenon. In previouswork with adult hepatocytes, the opposite (i.e., appearance ofAFP in short-term cultures) has been reported (11, 12). Of par-ticular interest is the report by Sirica et al. (27), who describeda liver cell culture system somewhat similar to ours. They foundthat adult rat liver cells could be grown on collagen gels. Thehepatocytes growing in such cultures also seemed to revert toa fetal phenotype as evidenced by appearance of AFP andGGTase. It is interesting that the changes in our cultures offetal

hepatocytes appeared to be in the opposite direction, the cellsexpressing a more adult phenotype as the cultures aged. Therat tail collagen used by Sirica et al. is almost exclusively typeI collagen, whereas the skin basement membrane contains typeIV collagen as well as noncollagenous basement membrane gly-coproteins such as laminin (28). In culturing of hepatocytes on

pigskin, scalpel blade slits must be made in the pigskin epi-dermis for the liver explants to grow (2, 19) and we have recentlyobtained evidence suggesting that basement membrane com-

ponents may facilitate -the attachment and growth of hepato-cytes. We have found that laminin enhances the adhesion ofcells from regenerating liver and that an increased expressionof laminin in the sinusoidal areas of the liver accompanies liverregeneration (29). It is possible that the type of matrix the livercells grow on influences their state of differentiation and thatour model supports normal ontogenetic development.

Our results also show that the culture system lends itself tostudy of hormonal control of differentiated functions of livercells. Addition of hydrocortisone to the medium prolongs AFPproduction (and supresses albumin production) but, at times,both products are synthesized at high levels. Thus, the AFP andalbumin genes operate independently, and maturation need notoccur for all genes at the same time.The effects of hydrocortisone on AFP and albumin produc-

tion seem to be specific and not a general effect on maturation.GGTase, like AFP, is a gene product typical offetal but not adulthepatocytes. Hydrocortisone had no effect of GGTase activity.Dissociation ofthe expression ofGGTase and AFP has also been

found in spontaneous hepatomas of mice (30). Unlike carcino-gen-induced hepatomas, the spontaneous hepatomas do notexpress increased amounts of GGTase but usually are positivefor AFP.We were somewhat surprised to find that hydrocortisone

prolonged the expression of AFP in-our cultures. The in vivoeffect of glucocorticoids on AFP synthesis has been found to bethe opposite. Administration of glucocorticoids, including hy-drocortisone, accelerated the decline of serum AFP levels innewborn animals (31). This effect is not due to increased rateof catabolism of AFP but seems to depend on a decrease in thecellular level of mRNA for AFP (32). The reason for this dis-crepancy between the in vitro and -in vivo activities remainsunclear. However, several target organs for hormone actionmay be involved in vivo; our system should reflect the directeffects of the hormone on the liver tissue.

Hydrocortisone has been found to induce increased quan-tities ofAFP in some cell lines derived from rat hepatomas (16,17). In our culture, once it had ceased in hydrocortisone-freemedium, AFP production was not restored by addition of hy-drocortisone. The synthesis ofAFP thus seems to be under tightregulation in this system. These long-term primary cultures ofnormal liver cells should be useful in answering questions onthe control of oncodevelopmental gene expression.

We thank Ms. Peggy Klett, Masato Miyashita, and Marsha Bell fortechnical assistance with the AFP and albumin assays, Florence E.Cardenas for technical assistance with histochemical analyses, PauletteMelfi and Paul Gumerlock for cell culture aspects, and Ms. Patty Saxenfor manuscript preparation. This work was supported by National Can-cer Institute Grants 1 ROI CA 25392-01 (A. E.F.), CA 27460 and CA27455 (E.R.), and CA 21967-03 to Dr. William H. Fishman (R.H.K.).

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