PCC4azal teratocarcinoma stem cell differentiation in culture

DEVELOPMENTAL

BIOLOGY

75, 78-92 (1980)

PCC4azal Teratocarcinoma

Stem Cell Differentiation

I. Biochemical CECILIA

W.Lo’

in Culture

Studies

AND NORTONB. GILULA

The Rockefeller University, 1230 York Avenue, New York, New York 10021 Received April 23, 1979; accepted in revised form August 2, 1979 PCClaxal embryonal carcinoma cells were observed to spontaneously differentiate under defined culture conditions to endoderm-like cells and subsequently to giant cells. This differentiation was examined by determining the specific activities of several enzymes in the stem and endoderm-like cell populations. With differentiation, the level of alkaline and acid phosphatase activities remained unchanged, plasminogen activator specific activity increased fivefold, and lactate dehydrogenase (LDH) specific activity decreased to 40% of its original level. Isozyme analysis revealed a shift of the LDH isozymes toward LDHl with the appearance of LDH2 for the fist time in the endoderm-like cells. The surface antigen &SEA-l was detected by indirect immunofluorescence on virtually all of the stem cells. However, the SSEA-1 antigen was not present on many of the endoderm-like cells, and it was completely undetectable on giant cells as assayed by immunofluorescence. The expression of H-2 antigen was examined in a similar manner using anti-H-2b antiserum; this antigen was not detected on the stem, endoderm-like, or giant cells. Thus, there are defined biochemical changes that accompany the differentiation of PCC4axal stem cells in culture. INTRODUCTION

1974; Martin and Evans, 1975). Some of these lines consist of embryonal carcinoma cells which can differentiate to varying extents in culture. PCC4azal is an embryonal carcinoma cell line isolated by Jakob et al. (1973) that has been reported to be capable of differentiation to all three germ layers in uiuo (Jakob et al., 1973), but it exhibits a limited differentiation in culture (Sherman, 1975). In this study, two sets of culture conditions have been defined: one which allows the continuous maintenance of homogenous populations of PCC4azal embryonal carcinoma cells; and another which promotes their uniform differentiation to endoderm-like derivatives. The undifferentiated and differentiated cells were analyzed for the expression of various enzymatic activities and two cell surface antigens, SSEA-1 (Solter and Knowles, 1978) and H-2b.

Teratocarcinomas are tumors that consist of a mixture of proliferating stem cells and differentiated cells of various types. The stem cells, or embryonal carcinoma cells, resemble early embryonic cells in their ability to differentiate extensively, sometimes forming derivatives of all three germ layers (Stevens, 1967). The teratocarcinema is an attractive model system for studying mouse embryonic development since a transplantable mouse teratocarcinoma has been developed that can be easily cultured. The teratocarcinoma can be serially propagated in the mouse as a solid subcutaneous tumor or in the peritoneum as floating aggregates called embryoid bodies (Stevens, 1970; Pierce et al., 1959). Many cell lines have been isolated from both the solid tumor and the embryoid bodies (Kahan and Ephrussi, 1970; Rosenthal et al., 1970; Jakob et al., 1973; Lehman et al.,

MATERIALS

’ Present address: Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 82115.

Materials Dulbecco’s 78

0012-1606/80/030078-15$02.00/O Copyright All rights

0 1980 by Academic Press, of reproduction in any form

Inc. reserved.

AND

modified

METHODS

Eagle’s

medium

Lo AND GILULA

Teratocarcinoma Cell Differentiation-Biochemistry

(MEM) with 4.5 g glucose/liter; 10,000 units-10,000 pg penicillin G-streptomycin/ ml saline (Grand Island Biological Co., Grand Island, N.Y.); Falcon plastic tissue culture ware (Bioquest, BBL, and Falcon Products, Cockeysville, Md.); fetal bovine serum and Linbro multiwell plate (Flow Laboratories, Inc., Rockville, Md.); COZ incubator (National Appliance); 8% aqueous glutaraldehyde (Nz sealed) and 4% aqueous osmium tetroxide (Polysciences Inc., Warrington, Pa.); analytical grade tannic acid (Mallinckrodt, St. Louis, MO.); uranyl acetate (Fisher Scientific Co., Pittsburgh, Pa.); and p-nitrophenyl phosphate, NAD, NADH, sodium lactate, sodium pyruvate, phenazine methosulfate, tetranitroblue tetrazolium, and 2-amino-2-methyl-l-propanol (Sigma Chemical Co., St. Louis, MO.). Fibrinogen and iodinated fibrinogen were generously provided by Dr. S. Strickland of The Rockefeller University. The 129 SvSl mice were obtained from the Jackson Laboratory, Bar Harbor, Maine.

Cell Culture and Embryoid Body Growth The cell line PCC4azal was originally isolated by Jakob et al. (1973) and kindly provided by Dr. Frank Ruddle of Yale University. After obtaining these cells, they were passaged in viuo as a solid tumor and reisolated for cell culture. To repassage the cells in uiuo, approximately 5 X lo6 cells were injected subcutaneously into a 129 SvSl mouse. After 4 weeks, the tumor that was formed was excised, minced, and collagenase treated using the protocol of Evans (1972). The cell suspension derived from the tumor was plated, and subsequently a proliferating stem cell population was obtained. This reisolated population of PCC4azal was used for all the studies. The PCC4azal cells were grown in Dulbecco’s MEM with 10% fetal bovine serum and 50 units-50 pg/ml of penicillin-streptomycin. They were maintained in a 5% CO, incubator at 37°C and subcultured every 36 hr. The following subculture pro-

79

cedure was used. The culture was left at room temperature for 5 min and the cells were then gently pipetted off the dish and pelleted by a brief centrifugation in a tabletop centrifuge. The pellet was resuspended by gentle pipetting in fresh medium. The cell suspension was then counted in a hemacytometer and replated at 5 x lo5 cells in 10 ml of medium per loo-mm dish. For obtaining differentiation, the embryonal carcinoma cells were plated at 80,000150,000 cells in l-l.5 ml medium in 35-mm dishes. After 7 days, the medium was aspirated and fresh medium added. The cultures were then left for another 5-7 days, at which time terminally differentiated giant cells appeared. The embryoid bodies were provided by Dr. Leroy Stevens of the Jackson Laboratory, and they were originally derived from the tumor OTT6050 (Stevens, 1970). They were grown as an ascites tumor in the peritoneum of a 129 SvSl male mouse. Every 4-5 weeks a small amount of embryoid body cell aggregates was recovered from the ascites and subsequently injected into the peritoneum of a new mouse. Thus, this procedure allowed the continuous propagation of the tumor line. The embryoid bodies were harvested from the peritoneum of injected mice after sacrificing. A horizontal incision was made in the abdomen and the skin was peeled back, exposing the peritoneum. A small hole was cut in the peritoneal wall, then a Pasteur pipe containing Dulbecco’s phosphate-buffered saline was repeatedly introduced into the cavity to flush out and remove the tumor aggregates.

Light Microscopy For light microscopy, the cells were prepared by the same procedure that was used for electron microscopy (Lo and Gilula, 1980a), including the en bloc staining with uranyl acetate. After this procedure, the cells were examined and photographed (still attached on the petri dishes) with Kodak

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DEVELOPMENTALBIOLOGY

Plus-X film using a Zeiss Photomicroscope II. Cell Lysates Cells were harvested from petri dishes by gentle pipetting and pelleted by a brief centrifugation in a clinical tabletop centrifuge. The medium was aspirated and the cells were resuspended in Dulbecco’s phosphatebuffered saline (PBS: KCl, 2 g/liter; KHzPO+ 2 g/liter; NaCl, 8 g/liter; Na2HP04. 7Hz0,2.16 g/liter). They were washed three more times with PBS and then lysed with 0.5% Triton X-100 in PBS at 4°C. To obtain a homogeneous suspension, the cell lysate was sonicated for approximately lo-20 set with a Model W185 sonifier cell disruptor (Heat Systems-Ultrasonics Inc., Plainview, N.J.). It was found that the LDH and acid phosphatase activities were extremely unstable when stored at -20°C; therefore, all enzyme assays were conducted within 4-8 hr after harvesting the cell lysates. Protein content in the cell lysates was determined by the method of Lowry et al. (1951).

Enzyme Assays Alkaline and acid phosphatases were assayed according to a standard spectrophotometric procedure in which the hydrolysis of p-nitrophenyl phosphate to p-nitropheno1 was monitored. For alkaline phosphatase, the assay was conducted at 37°C for lo-30 min in 0.09 M 2-amino-2-methyl-lpropanol buffer (pH 10.5) with 8 mM pnitrophenyl phosphate. For acid phosphatase, the assay was conducted at 37°C for 30-45 min in 0.15 M sodium acetate buffer (pH 5.0) with 8 m&f p-nitrophenyl phosphate. The reaction was terminated by adding an equal volume of 1.2 N NaOH, and the absorbance was measured at 410 nm in a Gilford spectrophotometer (Gilford Instrument Laboratories, Inc., Oberlin, Ohio). The specific activity of alkaline or acid phosphatase is defined as micromoles per minute per milligram of protein (units/mg). Lactate dehydrogenase (LDH) was

VOLUME 75,198O

measured with a spectrophotometric assay in which the conversion of NADH to NAD was monitored at 340 nm. The reaction mixture contained 0.2 M Tris-HCl (pH 7.5) with 6.6 milf NADH and 30 mM sodium pyruvate. The reaction was carried out at 37°C and the initial rate of decrease in absorbance at 340 nm was measured. The specific activity of LDH is defined as micromoles per minute per milligram of protein (units/mg). The K,, Michaelis-Menten constant, of the LDH activity was determined using this same assay procedure, except that the concentration of pyruvate was varied (see Fig. 5). Plasminogen activator activity in the culture medium (secreted) and in cell extracts was assayed using published procedures (Unkeless et al., 1973; Strickland and Beers, 1976). ‘251-Fibrin-coated multiwell dishes (Linbro) were incubated with the medium or cell extracts in the presence of plasminogen (or in plasminogen-depleted serum in controls). Each well contained approximately 100,000 cpm, and aliquots of the reaction mixture were analyzed for the release of ‘251-soluble polypeptides in a Packard Autogamma spectrometer. The plasminogen activator activity is expressed as cpm released by the sample per total cpm released by 0.25% trypsin. The reaction mixture contained 5-25 ~1 of sample, 250 ~1 of 0.1 M Tris (pH 8.1), and 2.5% acidtreated fetal bovine serum as the source of plasminogen. The reaction was conducted at 37°C for 3-5 hr for the culture medium and for 18 hr for cell extracts. Plasminogen activator activity was also measured by directly plating cells onto fibrin-coated dishes in medium with acidtreated serum. Aliquots of the medium were removed at various time intervals and counted. The activity is expressed as the percentage of total trypsin-releasable counts. Plasminogen-depleted serum and acidtreated serum were prepared according to published procedures (Deutsch and Mertz, 1970; Strickland et al., 1976).

Lo AND GILULA

Teratocarcinoma

LDH Isozyme Cell lysates were analyzed for the distribution of LDH isozymes using a slightly modified version of the method of Dietz and Lubrano (1967). Briefly, cell extracts were electrophoretically separated on 6.6% (w/v) polyacrylamide tube gels and the gels were then incubated with lactate, NAD, phenazine methosulfate, and tetranitroblue tetrazolium for the detection of LDH activity. The presence of LDH activity was indicated by the appearance of a purple reaction product, and the gels were subsequently scanned in a Gilford spectrophotometer at 660 nm using a linear transport attachment (Model 2410-s).

Cell Differentiation-Biochemistry

81

lumination. Photographs were recorded on Tri-X film and processed with Acufine. Additional controls in which cells were incubated with only anti-SSEA-1, anti-H-2b, or FITC-conjugated anti-mouse total globulin antiserum were processed in parallel for each cell type examined. RESULTS

PCC4azal stem cells grow in tightly packed “epithelial nests” with the basic morphology ascribed to embryonal carcinoma cells, the undifferentiated stem cells of the teratocarcinoma (for review, see Martin, 1975) (Fig. la). These cells are small, with a high nuclear to cytoplasmic ratio. Each nucleus contains a prominent nucleolus. The cells in a typical field in Immunofluorescence these cultures are morphologically homogeneous (Fig. la). Anti-SSEA-1 antiserum was generously The stem cells multiply with a doubling provided by Dr. Davor Solter of the Wistar Institute. The antiserum was diluted 1:lOOO time of 8-10 hr, and a strict and frequent passaging schedule is vital for maintaining with Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bo- the PCC4azal cells in their stem cell state; vine serum and frozen in small aliquots at delay of passaging or plating of a higher initial density invariably results in a mixed -80°C. Anti-H-2h antiserum was generously provided by Dr. B. Benacceraf of culture containing varying amounts of stem Harvard University, and it was used at a 1: cells and cells of different morphology. The 36-hr passaging schedule used in this study 20 dilution in complete medium. The fluoallows the continuous propagation of a pure rescein isothiocyanate (FITC)-conjugated stem cell population as judged by light mirabbit anti-mouse total globulin was purcroscopy. chased from Cappel Laboratories, Inc. To promote uniform differentiation, (Cochranville, Pa.), and used at a 1:40 diplated stem cells are allowed to grow withlution in complete medium. For immunofluorescent staining, all out passaging for a week. During this time washes and incubations were conducted at cell death occurs as the remainder of the cells undergoes a series of morphological 4°C. The cells were first washed 7 times At the end of 6 or 7 days, with Dulbecco’s MEM containing 10% transformations. heat-inactivated fetal bovine serum and the culture is fed with fresh medium and then incubated with either anti-SSEA-1 an- the cells further differentiate. This induction is irreversible and results in the unitiserum, anti-H-2h antiserum, or a nonimof the entire stem cell mune mouse serum for 10 min. The cells form transformation were then washed 7 times as above and population first to endoderm-like cells and then to giant cells (Fig. If). It occurs over incubated with FITC-conjugated rabbit a period of 11 to 14 days, and it can be anti-mouse total globulin for an additional separated into three morphologically dis10 min. After the final incubation, the cells were washed 10 times and immediately ex- tinct stages: Stage 1 is identified between amined for fluorescent staining with a Zeiss days 4 and 6; stage 2 between days 5 and 7; and stage 3 between days 8 and 14. microscope equipped with fluorescent epiil-

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DEVELOPMENTAL BIOLOGY

VOLUME 75.1980

FIG. 1. Phase-contrast light micrographs of PCC4azal cell differentiation. The bars in a and c represent 50 pm. See text for complete description. (a) Rapidly proliferating stem cells or embryonal carcinoma cells. X 200. (b) Cell elongation, stage 1 of differentiation. X 200. (c) Cell enlargement, early stage 2 of differentiation. X 320. (d) Cell enlargement, late stage 2 of differentiation. x 320. (e) Giant cell formation, early stage 3 of differentiation. x 200. (0 Giant cell formation, late stage 3 of differentiation. x 200.

Stage 1: Cell Elongation The first detectable change in these cells upon promotion to differentiation is a change in cell shape. The cells are elongated and sometimes more spread out at one end, thus giving them a triangular shape (Fig. lb). Accompanying this shape change is the appearance of phase retractile ridges that outline entire cell borders. Cell death as

indicated by unattached cells and debris is most prominent at this stage.

Stage 2: Cell Enlargement With time, the initially elongated cells enlarge and are arranged in a polygonal packing (Figs. lc and d). The previously refractile cell borders narrow to thin lines that are heavily stained by tannic acid.

Lo AND GILULA

Teratocarcinoma

During this stage, multinucleated cells and cells with micronuclei are present for the first time and the nuclear to cytoplasmic ratio is decreased. Cells of this stage and stage 1 are referred to as endoderm-like (see Discussion and Lo and Gilula, 1980a).

Stage 3: Giant Cell Formation The differentiating cells greatly increase in size so that some cells are more than 250 pm in length. These cells are distinctively fibroblast-like. The nucleus of these giant cells is larger than an entire embryonal carcinoma stem cell (compare Figs. la and f). The larger giant cells (Fig. If) appear later in time than the smaller ones (Fig. le), and they appear to be derived from them. Most of the organelles of the giant cells are sequestered around the perinuclear region. The cytoplasm contains many stress fibers, some of which extend for at least 200 pm in the larger cells. As a result of an increase in cell motility, the close cell-to-cell contact maintained thus far is disassembled, and isolated cells are frequently located at a distance from their parent clones. Ruffled membranes and pseudopod extensions are apparent at the cell periphery. The giant cells can be maintained in culture for several more weeks, during which time there are no further detectable changes in size or morphology.

Enzyme Activities of Embryonal Carcinoma Cells and Endoderm-like Cells To explore the possibility that these striking morphological changes observed in the PCC4azal cell cultures might be accompanied by biochemical changes, the activities of several enzymes were measured in the stem cells and also in the stages 1 and 2 endoderm-like cells.’

Alkaline Phosphatase Several studies have suggested that alkaline phosphatase activity decreases with ’ In all tables and figures, stem refers to stem cell extracts and endo refers to endoderm-like cell extracts.

83

Cell Differentiation-Biochemistry

embryonal carcinoma cell differentiation (Bernstine et al., 1973; Martin and Evans, 1975), while more recent studies have shown either no change (Wada et al., 1976) or an increase in enzyme activity with differentiation (Strickland and Mahdavi, 1978). Both the alkaline and the acid phosphatase activities of stem and endodermlike cells have been determined . Measurements of stem and endodermal cell extracts showed no significant differences in the specific activities of either of these two enzymes (Table 1). This is more clearly illustrated by determining the ratio of the specific activity of alkaline phosphatase to the specific activity of acid phosphatase; this ratio remains unchanged with endoderm formation.

Plasminogen Activator Activity Since it has been reported that plasminogen activator activity appears in the growth medium upon differentiation of embryonal carcinoma cells (Strickland and Sherman, 1976; Topp et al., 1976; Linney and Levinson, 1977), the plasminogen activator activity of the PCC4azal stem and endoderm-like cells was measured. Initial measurements revealed no detectable plasminogen activator activity in the medium of stem cell cultures, while the medium from cultures containing endoderm-like cells contained large amounts of activity when assayed under identical conditions (data not shown). However, since endoderm-like cultures contain more cells, this difference may at least partially reflect (1) the difference in cell number in the two TABLE 1 ALKALINE AND ACID PHOSPHATASE ACTIVITY OF PCC4axal CELLS Cdl Specific activity 1 Specific (pmoles/min/mg) activity type /~___ --- (alk Alk Pase ~ Acid Pase Pase/ Iacid Pase Stem Endo

0.097 + 0.01 (5)” I 0.021 + 0.002 (5) 0.083 + 0.02 (12) IO.018 + 0.002 (12)

4.63 4.68

n The number in parentheses is the number of separate cell preparations assayed.

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DEVELOPMENTAL

BIOLOGY

cultures and (2) the limit of the sensitivity of the assay itself. To determine whether the stem cells produce any plasminogen activator, they were plated directly into dishes which were precoated with ‘251-fibrinogen. This is a more sensitive assay for plasminogen activator activity (Strickland, personal communication) in which the release of radioactive counts into the growth medium directly reflects the amount of plasminogen activator activity present. The PCC4azal stem cells assayed in this manner express detectable plasminogen activator activity (Fig. 2a). To determine whether this is peculiar to this particular embryonal carcinoma cell line, two other embryonal carcinoma cell lines, PCC3 and F9, were similarly assayed and plasminogen activator activity was again detected in both lines (Figs. 2b and c). In all cases, no fibrinolytic activity was detected in embryonal carcinoma cells plated in parallel but without plasminogen. In addition, the possibility that the fibrinogen on the dishes might somehow induce the expression of plasminogen activator activity in these cells was examined by plating equal numbers of PCC4azal stem cells with medium containing plasminogen-depleted serum in (1) dishes coated with 1251-radiolabeled fibrinogen, (2) dishes coated with unlabeled fibrinogen, and (3) dishes that were not coated. After 36 hr of incubation, the medium was removed from each set of dishes and the entire contents were assayed for plasminogen activator activity. The levels of plasminogen activator activity recovered from the medium of ‘251-fibrinogencoated, fibrinogen-coated, and uncoated dishes were identical (Fig. 3). No plasminogen activator activity was detected in cells plated in a parallel manner but in the absence of plasminogen. Therefore, the plasminogen activator activity detected from stem cells plated onto the 1251-fibrinogencoated dishes does not result from induction by the coated fibrinogen, but rather it must be an intrinsic activity of the plated cells.

VOLUME

75.1980

,Or

(a) PCC4azal 16K

60

40

8K 4K

20

2K /,A t 0 12

I6

24

30

36

o"T-2

I6

24

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/ eoF/

, 66K

Hours FIG. 2. Plasminogen activator activity in three embryo& carcinoma cell lines. Embryonal carcinoma cells in Dulbecco’s MEM with 10% fetal bovine serum were plated directly into ‘251-fibrinogen-coated wells of Linbro plates and maintained at 37°C in a 5% CO2 incubator. The release of soluble counts into the medium was followed over time, and the number of cells (X 103) plated per well is indicated by the number to the right of each curve. The cell lines examined were: (a) PCClazal, (b) PCC3, and (c) F9.

The level of plasminogen activator activity was compared in the stem cells and in the endoderm-like cells by preparing cell lysates of each cell population that were assayed in parallel. Compared to stem cells, the endoderm-like cells contained approximately five times more plasminogen acti-

Teratocarcinoma

Lo AND GILULA

LC

I

FIG. 3. Effect of plasminogen-coated substratum on plasminogen activator activity of PCC4azal embryonal carcinoma cells. PCC4azal embryonal carcinoma cells were plated in parallel into uncoated Linbro wells (L) or Linbro wells coated with ‘2”I-fibrinogen (I) or unlabeled (cold) fibrinogen (C). Cells were plated in Dulbecco’s MEM with 10% plasminogen-depleted and acid-treated fetal bovine serum at 6.4 x IO4 cells per well. After 36 hr of incubation at 37°C in a 5% CO, incubator, the medium from each well was collected and any cells or debris were removed by a brief centrifugation. Each harvested sample was then placed in a ‘“‘I-fibrinogen-coated Linbro well (that had been activated), and acid-treated fetal bovine serum was added to each well as a source of plasminogen. The Linbro plates were then incubated at 37°C for 48 hr, after which the soluble counts released from each sample were measured.

vator activity 4).

per milligram

Lactate Dehydrogenase

85

Cell Differentiation-Biochemistry

of protein (Fig.

Activity

The stem cells grow optimally in a medium with a high concentration of glucose. During the 36 hr of growth in culture between passages, the medium rapidly becomes acidic due to the accumulation of lactate. However, the endoderm-like cells and giant cells probably have a reduced level of lactate production, as indicated by the slower rate at which their growth medium becomes acidic. In addition, direct measurements with living cells have shown that endoderm-like cells have twice the rate of 02 consumption as compared to stem cells and also are elevated in CO2 production (unpublished observations). These observations suggest that there is a change in the glucose metabolism of these cells upon differentiation, whereby perhaps more of the glucose, instead of being oxidized to lactate, is metabolized completely to COZ.

L

:

J Enda

--

FIG. 4. Plasminogen activator activity in PCC4azal stem and endoderm-like cells. PCC4azal stem and endoderm-like cell lysates were assayed in parallel for plasminogen activator activity as determined by the solubilization of ‘*“I-fibrin. The activities (percentage substrate solubilized) were normalized by the protein content in each sample. The specific activity of stem cell lysates was arbitrarily defined as 1, and the relative specific activity of endoderm-like cell lysates was derived by dividing the specific activity of endoderm-like cell lysates by the specific activity of stem cell lysates.

A possible key enzyme in the regulation of glucose metabolism is lactate dehydrogenase (LDH), a tetrameric enzyme composed of various combinations of the two subunits, M and H, subunit M having a higher affinity for pyruvate. Five isozymes composed of the five possible combinations of the two subunits (LDH5 containing pure subunit M to LDHl containing pure subunit H) are found in varying proportions in different tissues, with a predominance toward LDH5 in highly glycolytic tissue such as skeletal muscle. Therefore LDH activity was determined in the PCC4azal cells before and after differentiation. LDH activity decreases with differentiation to 40% of the specific activity of stem cells in the endoderm-like cells (Table 2). Determination of the K, of the LDH activity of the stem and endoderm-like cells by Lineweaver-Burk reciprocal plot analysis showed that there is no significant change in the K,,, of the LDH activity with differentiation (Table 2). In order to directly analyze the distribution of LDH isozymes in the stem and endoderm-like cells, LDH isozymes in the cell lysates were separated by polyacrylamide gel electrophoresis and the location of the LDH activity in the gels was visual-

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antibody (hybridoma) generated against the embryonal carcinoma cell line F9, is SpAfiC Specific activity expressed by embryonal carcinoma cells Kl” activity Cell tYPe (fimoles/min/mg) (endo but not their differentiated derivatives (SolLDH/ ter et cd., 1979.) This antigen is also present stem LDH) on the cells of the mouse embryo at specific 3.61 f. 0.04 (5)* 306 x W3 Stem stages, and hence has been labeled stageo.4 342 x 10-3 Endo 1.45 + 0.25 (12) specific embryonic antigen 1, SSEA-1. Usn Michael&Menten constant expressed as microing this monoclonal antiserum (antiSSEAmoles per liter of pyruvate. 1) and FITC-conjugated rabbit anti-mouse * The number in parentheses is the number of septotal globulin, PCC4azal stem cells, endoarate cell preparations assayed. derm-like cells, and giant cells were examized by a purple reaction product (as de- ined for the presence of the SSEA-1 antigen scribed in Materials and Methods). The by immunofluorescence. Most stem cells stem cells have three LDH isozymes, LDHB, were heavily stained (positive cells) (Fig. LDH4, and LDH3, while the endoderm-like 6a); however, a very small percentage of the cells have four isozymes, including a new stem cells was not stained at all (negative isozyme not present in stem cells, LDHz cells). These negative cells were usually (Fig. 5a). For comparison, the LDH activi- found side by side within clones of positive ties of embryoid bodies (collected from the cells. No clones of completely negative cells ascities of 129 SvSl mice), fetal bovine se- have ever been seen. The endoderm-like rum, and mouse serum were similarly ana- cell cultures have a large proportion of unlyzed. The embryoid bodies have four iso- stained cells. The negative endodermal cells zymes, with only a small amount of LDHz were usually the larger, more differentiated (which is not clearly visible in the gel in endoderm-like cells (Figs. lc and d and Fig. Fig. 5a). Fetal bovine serum contains five 6), and both completely negative clones as isozymes, with a predominance toward well as clones containing both positive and LDH1, while mouse serum has five bands, negative cells were observed in the endowith a predominance toward LDH5. The derm-like cell cultures. No completely pospattern observed in the stem and endo- itive clones were observed. In contrast, no derm-like cells was distinct from that of the giant cells were ever detectably stained by fetal bovine serum and cannot be due to this antiserum. contaminating fetal bovine serum in the The expression of H-2 antigen by the cell extracts. Densitometric scans of the stem, endoderm-like, and giant cells was gels show a marked shift of the LDH iso- also examined by immunofluorescence uszymes toward LDHl upon differentiation to ing anti-H-2b antisera and FITC-conjuendoderm (Fig. 5b and Table 3), resulting gated rabbit anti-mouse total globulin as in a decreasing proportion of LDH activity described above. It has been reported that in LDH5 and an increasing proportion of teratocarcinoma cells, similar to early emLDHl and LDH3 activity, as well as the bryonic cells, do not express H-2, but as new appearance of LDH2. This shift in iso- development proceeds the expression of Hzyme distribution may possibly reflect a 2 becomes detectable (Stern et al., 1975; change in glucose metabolism in the Jacob, 1977). No staining was observed in PCC4azal cells as they differentiate to en- any of the PCC4azal cells, including stem, doderm-like cells. endoderm-like, and giant cells (data not shown), with anti-H-2b antiserum. Cell Surface Antigen TABLE

2

LDH ACTIVITY OF PCC4azal CELLS

It has been reported that a specific cell surface antigen, as defined by a monoclonal

DISCUSSION

The

PCC4azal embryonal carcinoma

Lo

AND

GILULA

Teratocarcinoma

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Cell Differentiation-Biochemistry

LDH, LDH4 LDH, LDH, LDH,

08

2

06

06

04

02

10 cm

i

0 10

cm

I

20

0

0

10

20

cm

FIG. 5. LDH isozyme analysis by polyacrylamide gel electrophoresis. The LDH isozymes in the following samples were separated by polyacrylamide gel electrophoresis and the LDH activities subsecmently identified by a colored reaction product (as described in Materials and Methods): (1) stem cell lysates; (2) endoderm-like cell lysates; (3) embryoid body cell lysates; (4) mouse serum; (5) fetal bovine serum. 126.6 units of LDH activity was loaded into 1, and 58.8 units of LDH activity was loaded into 2. Densitometric scans of gels 1, 2, and 4 (at 660 nm) are shown below.

cells can be maintained as stem cells or they can be triggered to undergo a transformation to endoderm-like cells and then to giant cells. The transformation in cell morphology observed in this study is accompanied by changes in the specific activity of LDH, the LDH isozymic distribution, and the plasminogen activator specific activity and by the loss of the SSEA-1 antigen on the cell surface. These concomitant

changes in morphology and enzyme and surface properties suggest that a specific differentiation process is induced in this system. It is with these criteria in mind that this process of endoderm and giant cell formation is referred to as a differentiation process in this and subsequent studies (Lo and Gilula, 1980a, b). The stages 1 and 2 differentiated cells were called endoderm-like in this study

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DEVELOPMENTAL BIOLOGY TABLE

3

DISTRIBUTION OF LDH ISOZYMES IN PCC4azal CELLS AND MOUSE SERUM” Percentage

LDHs LDHd LDH3 LDHz LDH,

Stem

Endo

68.41 29.56 2.03

55.3 34.5 9.66 0.55

of total activity Mouse serum

Endo/ stem’

37.10 29.72 29.45 3.42 0.32

0.81 1.17 4.76 m

a Based on the area under each peak of LDH activity in the densitometric scan of the polyacrylamide gels (Figs. 5a and b). b Percentage of total activity of endoderm-like cells divided by percentage of total activity of stem cells.

since, in other studies, cells of similar morphology in teratocarcinoma cell cultures have been called endoderm (Sherman et al., 1976; Strickland and Mudhavi, 1978). They have been labeled as such by others because they are believed to be related to yolk sac endoderm of the mouse embryo. The formation of giant cells by teratocarcinema stem cells has also been observed in other studies: in PCC3 cultures treated with hexamethylenebisacetamide (Dubois et al., 1978); in rare instances, in plated PCC4azal cell aggregates (Sherman, 1975); and in postconfluent cultures of PCC3 cells, in which the giant cell is one of many differentiated cell types that is spontaneously formed (Nicolas et al., 1976). In this study the ordered progression of stem cell differentiation to endoderm-like cells and then to giant cells has been observed. The changes in the specific activity of LDH and the distribution of LDH isozymes upon differentiation are consistent with a decrease in aerobic glycolysis, which would

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account for the decrease in lactate production by endoderm-like and giant cells. The distribution of LDH isozymes in the embryoid bodies is comparable to that in the endoderm-like cells formed in culture, but the proportion of LDHz is lower. The embryoid bodies used in this study consist predominantly of both stem cells and endoderm-like cells (Lo and Gilula, 1980b), and therefore they would be expected to have an isozymic profile in between the LDH isozymic distributions of pure stem cells and pure endoderm-like cells. The secretion of plasminogen activator activity into the culture medium by endoderm-like cells was first reported by Strickland et al. (1971) and has since been detected in embryoid body cultures and other differentiating teratocarcinoma cell cultures (Topp et al., 1976; Linney and Levinson, 1977; Strickland and Mahdavi, 1978). The observation here of increased plasminogen activator production in endoderm-like cells is consistent with the findings in these previous studies. The plasminogen activator activity detected in PCC4aza1, F9, and PCC3 stem cell cultures is probably endogenous to stem cells and not due to the presence of contaminating endoderm-like cells. However, it must be noted that the level of plasminogen activator activity in the stem cells is low compared to that in the endoderm-like cells. Alkaline phosphatase, which has been reported to change with embryonal carcinoma cell differentiation and, in fact, to be completely absent in endoderm-like cells (Bernstine et al., 1973; Martin and Evans, 1975), is expressed in both PCC4azal stem and endoderm-like cells at the same level.

FIG. 6. Immunofluorescent staining of PCC4azal cells with the monoclonal antibody SSEA-1. The presence of SSEA-1 antigen on the cell surface of the PCC4azal cell was examined by immunofluroescence (as described in Materials and Methods). a, c, e, and g are phase images of the specific regions examined with epifluorescence in b, d, f, and h. (a, b) Stem cells are uniformly stained (c, d) Some endoderm-like cells are stained, while others are not stained at all. The intensity of staining also is variable. (e, f) The more differentiated or larger endodermlike cells are not stained. The bright spots are due to debris which nonspecifically traps the secondary antibody label (FITC rabbit anti-mouse total globulin). (g, h) The giant cells are not stained. The bar in a represents 24.4 pm.

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6. a-h.

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It should be indicated that most reports of a decrease in alkaline phosphatase activity involved the analysis of mixed cell populations in which many different differentiation processes were ongoing, or were based solely on histochemical staining. These differences in methods and/or materials may account for the presistence of alkaline phosphatase observed in this study. The SSEA-1 antigen disappeared as PCC4azal stem cells differentiated to endoderm-like cells and then to giant cells. Likewise, F9 stem cells which have been induced to differentiate by retinoic acid also lose this antigen as they differentiate to endoderm-like cells (Solter et al., 1979). The SSEA-1 antigen has been observed to appear at the eight-cell stage of the mouse embryo and to disappear from the trophectoderm with further development (Solter and Knowles, 1978). The inner cell mass was also observed to express SSEA-1, and the outer endoderm layer was “mostly positive” (Solter and Knowles, 1978). Currently, there is no evidence to suggest whether the loss of SSEA-1 antigen from the endoderm-like cells and giant cells correlates with events in the early mouse embryo; however, it has been suggested that the endoderm-like cells observed in the teratocarcinoma system, either in culture or in the tumor in Go, are identical to the visceral or parietal endoderm of the yolk sac of the mouse embryo (Martin et al., 1977; Strickland and Mahdavi, 1978). There is a small number of embryonal carcinoma cells which do not express SSEA-1 as indicated by immunofluorescence. The inability to detect SSEA-1 on these cells may be due to shedding or stripping of the antigen during passaging of the cells. This is based on the observation that the older stem cell cultures have fewer of the nonstaining cells as compared to a more recently plated culture. Similar observations have been made on F9 cells (Solter et al., personal communication). In the future, these immunofluorescence observations can be clarified by direct immunochemical

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characterization of the SSEA-1 antigen on these cells. The absence of H-2 antigen in the PCC4azal cells is in direct agreement with studies in which PCC3 cells were induced to form giant cells that also had no detectable expression of H-2 before or after differentiation (Dubois et al., 1978). In addition, the previous studies of Stern et al. (1975) showed that the endodermal cells of embryoid bodies also did not express H-2. The cell death which is observed during differentiation in this culture system is not unlike the cell death which accompanies the differentiation of embryoid bodies plated in culture (unpublished observations). It occurs randomly among all the clones in a culture. Since the embryonal carcinoma cells are plated as single cells, the process of cell death probably cannot be directly related to either the selective survival of contaminating differentiated cells in the culture or the selection for a subset of the embryonal carcinoma cells with dissimilar differentiation potential. Since endoderm formation by embryo& carcinoma cells is elicited by not changing the culture medium, the induction of this differentiation process may be critically dependent on the concentration of specific metabolites in the medium. In addition, if this induction is also dependent on the cell cycle, then one possible explanation for the observed cell death is that cells would only differentiate if they are at the responsive phase(s) of the cell cycle when the culture medium becomes inductive or otherwise die if they attempt to continue proliferating as stem cells. At present, the significance of this phenomenon is not understood, but in many differentiation systems in uiuo, development is often accompanied by specific cell death (Ghicksmann, 1951; Saunders, 1966; Hamburger, 1975). An understanding of the induction process in this system which leads to the formation of endoderm and giant cells may provide some insight into the variables that are involved in regulating the differential

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gene expression of early embryonic stem cells. Since this system will differentiate in culture in a homogeneous manner to endoderm-like cells, it provides an opportunity for a thorough biochemical analysis of this induction process and the subsequent expression of cellular differentiation. We would like to thank Dr. Sidney Strickland for many helpful discussions and advice, especially with regard to the plasminogen activator determinations, and also for reviewing the manuscript. We would also like to thank Ms. Asneth Kloesman and Ms. Kathy Wall for help in the preparation of figures and Mrs. Madeleine Naylor for secretarial assistance. This research was supported by grants from the United States Public Health Service (HL 16507 and GM 24753) and The Rockefeller Foundation (RF 70095). N. B. Gilula is the recipient of a Research Career Development Award (HL 00110). REFERENCES BERNSTINE, E. G., HOOPER, M. L., GRANDCHAMP, S., and EPHRUSSI, B. (1973). Alkaline phosphatase activity in mouse teratoma. Proc. Nat. Acad. Sci. USA 70, 3899-3903. DEUTSCH, D. G., and MERTZ, E. L. (1970). Plasminogen purification from human plasma by affinity chromatography. Science 170,1095-1096. DIETZ, A. A., and LUBRANO, T. (1967). Separation and quantitation of lactic dehydrogenase isoenzymes by disc electrophoresis. Anal. Biochem. 20, 246-257. DUBOIS, P., EISEN, H., and JACOB, F. (1978). Effet de l’hexamethylenebisacetamide sur la differentiation de cellules de carcinome embryonnaire. C. R. Acad. Sci. Paris Ser. D T-286, No. 1, 109-111. EVANS, M. J. (1972). The isolation and properties of a clonal tissue culture strain of pluripotent mouse teratoma cells. J. Embryol. Exp. Morphol. 28, 163176. GL~~CKSMANN, A. (1951). Cell death in normal vertebrate ontogeny. Biol. Rev. (Cambridge) 26, 59-86. HAMBURGER, V. (1975). Cell death in the development of the lateral motor column of the chick embryo. J. Comp. Neurol. 160, 535-546. JACOB, F. (1977). Mouse teratocarcinoma and embryonic antigens. Immunol. Rev. 33, 3-32. JAKOB, H., BOON, L., GAILLARD, J., NICOLAS, J. F., and JACOB, F. (1973). Teratocarcinome de la sourisisolement, culture et proprietes de cellules a potentialites multiples. Ann. Microbial. (Inst. Pasteur) 124B, 269-282. KAHAN, B. W., and EPHRUSSI, B. (1970). Developmental potentialities of clonal in vitro cultures of mouse testicular teratoma. J. Nat. Cancer Inst. 44, 1015-1036. LEHMAN, J. M., SPEERS, W. C., DWARTZENDRULER, D.

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STERN,P. L., MARTIN,G. R., and EVANS,M. J. (1975). Cell surface antigensof clonal teratocarcinoma cells at various stages of differentiation. Cell 6, 455-465. STEVENS, L. C. (1970).The development of transplantable teratocarcinomas from intratesticulargrafts of pre- and postimplantationmouse embryos. Develop. Biol. 21, 364-382. STEVENS,L. C. (1967). The biology of teratomas. Advan. Morphogen. 6, l-32. STRICKLAND, S., and BEERS,W. H. (1976). Studies on the role of plasminogen activator in ovulation: In vitro response of granulosa cells to gonadotropins, cyclic nucleotides and prostaglandins. J. Biol. Chem. 251,5694-5702. STRICKLAND, S., and MAHDAVI,U. (1978). The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 15,393-403. STRICKLAND, S., REICH, E., and SHERMAN,M. L. (1976). Plasminogen activator in early embryogen-

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