HISTOCHEMICAL AND ULTRASTRUCTURAL ...HISTOCHEMICAL AND ULTRASTRUCTURAL CHARACTERISTICS OF A CELL...

J. Cell Sci. 50, 281-297 (1981) 281Printed in Great Britain © Company of Biologists Limited IQ8I

HISTOCHEMICAL AND ULTRASTRUCTURAL

CHARACTERISTICS OF A CELL LINE FROM

HUMAN BONE-MARROW STROMA

MICHEL LANOTTE1, TERENCE D.ALLEN1 ANDT. MICHAEL DEXTER1

1 Department of Experimental Haematology and * Department of Ultrastructure,Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington,Manchester M20 gBX, England

SUMMARY

Morphological, enzymic and antigenic data are presented regarding a human bone-marrowstromal cell line maintained for 10 months and subcultured weekly. The main characteristicsare a fibroblastoid morphology, diffuse growth in collagen gels, no colony formation in soft gelmedia, contact inhibition of growth and conversion to adipocytea when treated with hydro-cortisone. The cells are non-phagocytic and membrane Fc receptors (i.e. aggregated humanimmunoglobulin G receptors) are absent, but they show diffuse cytoplasmic non-specificesterase activity, a strong acid phosphatase reaction, and a negative immunofluorescence (directand indirect) against factor VIII antigen. Other cell lines also have been isolated and main-tained in culture and present similar characteristics. These cell lines are thought to be derivedfrom the acid-phosphatase-positive marrow stroma directly associated with bone trabecularmatrix and probably represent a component of the haemopoietic inductive microenvironment.As such, they may provide a useful tool for studies in vitro of cell interactions and regulatoryprocesses in the control of human bone-marrow haemopoiesis.

INTRODUCTION

All components of the haemopoietic system (bone marrow, spleen, thymus andlymph nodes) show a complex association of stromal and haemopoietic moieties andnumerous models have been described concerning the role of such stromal elementsin the regulation of haemopoietic cell proliferation and differentiation. For example,the haemopoietic inductive microenvironment (HIM) concept (Trentin, 1970) pro-poses that stem-cell differentiation is regulated at a local level and the environmentalmilieu determines the cell lineages produced; e.g. the microenvironment of themarrow favours granulopoiesis while the microenvironment of the spleen favourserythropoiesis (La Pushin & Trentin, 1977).

Results obtained by the long-term liquid bone-marrow culture method (Dexter &Testa, 1976; Dexter, Allen & Lajtha, 1977) also indicate the necessity for a functionalHIM for CFU-S proliferation and concomitant differentiation and proliferationof the haemopoietic lineages. Indeed, the main feature of this method is the initialestablishment of an adherent cellular environment containing epithelial and fibro-blastic-like cells as well as endothelial, phagocytic cells and large adipocytes (Allen &Dexter, 1976a). However, although the long-term culture system maintains stem-cell

!° CE.L 50

282 M. Lanotte, T. D. Allen and T. M. Dexter

proliferation and the production of differentiated progeny for many months, there isstill controversy as to which stromal cells are important regulatory components.Furthermore, little is known of the nature of the stromal/haemopoietic cell interac-tions or the inductive mechanisms involved. It has been reported (Friedenstein et al.1974, 1976) that cultured fibroblastic cells from rabbit or guinea-pig marrow willtransfer the microenvironment of their original haemopoietic tissues when trans-planted under the kidney capsule of autologous animals. However, the contributionof the donor stromal elements (versus migrating host cells) was not determined.Although the cellular nature of the HIM has not so far been resolved, recently, 2types of stromal cells have been differentiated in rodent bone marrow (Westen &Bainton, 1979): an alkaline-phosphatase-positive (Alk-Pase) reticulum cell and anacid-phosphatase-positive (Ac-Pase) cell associated with granulocytic and erythroidprecursors, respectively.

However, all of these studies discussed have been performed in rodents and rela-tively little information is available on the features of human bone-marrow stroma,although preliminary observations in human long-term cultures show that stromalelements play an important role (Toogood et al. 1980; Hocking & Golde, 1980;Gartner & Kaplan, 1980). In order to analyse further the relative importance of thedifferent stromal cell types and to characterize the various factors being produced, itseemed to us that the production of a cloned cell line would be of great benefit. Usingsuch cell lines it may be possible to recreate the environment necessary for haemo-poiesis and to analyse in vitro the various haemopoietic disorders based upon environ-mental or haemopoietic cells.

In this paper, we report morphological, enzymic and antigenic patterns of normalhuman bone-marrow stromal cell lines, which have now been maintained and sub-cultured weekly for 10 months.

MATERIALS ANp METHODS

Donors

Rib specimens were obtained from patients undergoing cardiac surgery. The patients hada normal haemopoietic status and had not been treated with radiation or chemotherapy.

Treatment of the specimens

The rib fragments were kept cold and dissected under sterile conditions. The bone wascarefully cleaned of muscle fragments and connective tissue. The rib bone was cut around theperiphery with scissors, then opened (this procedure avoids contamination of the marrow withextraneous cellular elements). Small pieces of haemopoieitic bone trabeculae were detachedwith forceps and teased in culture medium.

Conditions of culture

Liquid culturet. Large cell aggregates were removed from the cell suspension by sedimenta-tion at 1 g at room temperature for 3-5 min. A total of io* to io6 cells were cultured in plastictissue culture flasks (Falcon) containing 10 ml Fischers' medium (Gibco) supplemented withL-glutamine (2 ITIM), 20 % foetal calf serum (FCS) and antibiotics, and maintained at 37 °Cin an atmosphere of air+ 5 % CO2. In some cases horse serum was used in place of FCS.

Human marrow stromal cell line 283

For subculturing, the cultures were trypsinized immediately before confluency, and split1/4 or 1/10. Cell growth was estimated by counting on a haemocytometer. The effects of somegrowth promoters and inhibitors were tested: fibroblast growth factor (FGF, 10 fig), epidermalgrowth factor (EGF, 100 fig) were purchased from Collaborative Research Waltham, Mass.,carrageenan (Marine Colloids - Seakem G. Rex 8074), cholera toxin (Sigma), insulin (Wellcome,London) (Green, 1978).

Collagen cultures. Collagen gels were established as previously described (Schor, 1980;Lanotte, Schor & Dexter, 1981). Fragments of bone trabeculae were cultured on such gels in1 ml supplemented Fischers' medium. When specified, single-cell suspensions of stromal cellswere plated in the collagen gel matrix in order to evaluate colony formation. When necessary,cells were recovered by treatment with collagenase (c i mg/ml collagenase, Sigma) for i h at37 CC. For morphology, the collagen gels were dried and the cells stained in situ with May-Grunwald-Giemsa.

Transmission (TEM) and scanning (SEM) electron microscopy

The methods were as described previously (Allen & Dexter, 1976a). Briefly they are asfollows: for TEM the adherent layer was fixed in situ in 3 % glutaraldehyde in phosphate buffer,post-fixed with osmium tetroxide in the same buffer, dehydrated and embedded in situ inLuft's Epon. Sections were cut at right angles to the growing surface. For SEM the flaskswere fixed as above, and several regions of the growing surface removed with a warmed corkborer, critical-point dried from CO, using amyl acetate as the transitional fluid, and sputter-coated with 200 A of gold.

Test of phagocyte function

Phagocytic cells in the adherent cell populations cultured from human bone marrow wereevaluated employing 3 different techniques:

(1) Opsonized yeast (2 x 10' particles/cm1) was incubated in the cultures for 2 h at 37 °Cin medium supplemented with 15% non-heat-activated FCS (or supplemented with 5%guinea-pig complement, diluted 1/30, Gibco N.Y.).

(2) Sheep red blood cells (SRBC) fixed with glutaraldehyde.(3) Latex particles (1-5 fim) at 2 mg/ml in the culture medium.

The layers were washed twice after incubation, then fixed and stained.

Histochemical analysis of enzymic markers

Acid phosphatase and alkaline phosphatase activity. The Ac-Pase procedure used was basedupon Barka's method (Barka & Anderson, 1962). Cell layers were fixed and incubated in asolution containing naphthol AS-BI phosphoric acid and Fast Garnet GBC (Kaplow &Burstone, 1964). After 60 min incubation at 37 °C in the dark, the slides were washed indeionized water for 3 min. For Alk-Pase, cells were stained using naphthol AS phosphate andFast Blue BBN (Kaplow, 1955).

Naphthol ASD chloroacetate esterase. A modification of the method described by Moloney(Moloney, McPherson & Fliegelman, i960) was used. The staining solution was made bydissolving 10 mg naphthol ASD chloroacetate substrate in 1-6 ml dimethylsulphoxide (DMSO)and adding 12 ml distilled water,- 1 ml propylene glycol, 12 ml o-i M-barbiturate buffer(pH 7-4). Fast Garnet salt GBC was added to the solution, then it was filtered. The slideswere stained for 30 min at room temperature.

Naphthol ASD acetate esterase. The staining solution contained: 10 mg naphthol ASDacetate dissolved in 1 ml acetone and 1 ml propylene glycol. The substrate solutions weremixed progressively with o-i M-phosphate buffer, propylene glycol (2%) (20 ml). Fast Bluesalt was added and the slides stained 30 min at 37 °C.

a-Naphthyl acetate esterase. The incubation solution was made as follows: 10 mg a-naphthylacetate was dissolved in 0-25 ml acetone and added to 20 ml of 0-15 M phosphate buffer (pH7-4) then shaken for 1-2 min. The solution was filtered directly onto the wet slides and incuba-ted for 15 min at room temperature. The slides were washed with distilled water.


Factor VIII antigen immunofluorescence labelling

Goat anti-rabbit antisera and rabbit anti-human factor VIII antigen antisera were obtainedfrom Miles (Immunochemicals and Miles Lab. Ltd, UK).

Stromal cells cultured on glass coverslips were washed twice with PBS. Cells were incubatedwith labelled or unlabelled rabbit antifactor VIII antigen antisera after 30 min fixation withacetone (4 °C). Slides were incubated in unlabelled antisera, and then incubated (indirectmethod) with goat anti-rabbit fluorescent antisera for 30 min at room temperature, washedand mounted in glycerophosphate. Similarly treated human umbilical-cord endothelial cellswere used as a positive control.

Incorporation of ^Cjpalmitic acid into cellular lipids; cellular triglyceride extraction andsilica gel thin-layer chromatography

Lipids were labelled with 14C-uniformly-labelled palmitic acid (403 mCi/mmol) in hexanesolution (Amersham, U.K.). Cultures were incubated in 5 ml of medium containing 0-5 pd[14C]palmitic acid at 37 °C; 30 min later, then subsequently, at hourly intervals, 50-/4I sampleswere taken from the incubation medium for an evaluation of isotope clearance. After the periodof incorporation, the cultures were washed. The lipids were extracted as described earlier(Green & Kehinde, 1974).

Binding of protein A-{SRBC) by aggregated human IgG

The culture medium was discarded from the culture flask and the adherent cells washedtwice by incubation with phosphate-buffered saline (PBS) for 15 min. One ml of aggregatedhuman IgG (200/ig/ml or 1 mg/ml) in solution in PBS supplemented with 10% FCS (Papa-michail et al. 1979) was added to each 25 cm* culture flask and incubated for 30-45 min at4 °C. The incubation mixture was decanted and the layers were washed twice with PBS(each was followed by 15 min incubation at room temperature). Two ml of a protein A-coatedSRBC suspension (2 % cells, v/v) was added to each flask and incubated without disturbancefor 45 min at room temperature. The supematants were gently discarded and replaced by aPBS/FCS solution and the number of cells forming rosettes was determined at x 60 magnifica-tion.

Irradiation experiments

Cells in suspension in serum-free medium were chilled to 4 °C on ice and submitted toirradiation using a 18'Cs source delivering a dose of 400 rad per min. After irradiation cellswere appropriately diluted in complete culture medium and immediately plated. When cellswere cultured at low concentration a feeder of lethally irradiated cells (iol cells/cm1, 1500 rad)was used to promote colony growth.

RESULTS

Isolation of a pure stromal cell population

Primary cultures of bone-marrow cells were established using various concentra-tions of cells from io* to 5 x io6 per culture (10 ml). The cells were incubated for 4days at 37 °C and subsequently all the floating cells were removed. At the low cellconcentration small clusters of cells were observed. The cultures were fed with freshmedium and incubated in the same conditions. After 2 weeks the cultures wereexamined with a phase-contrast microscope. Where low numbers of cells wereoriginally plated there was little or no growth of stromal cells. At the higher concen-tration a confluent layer of fibroblast-like cells was formed. We selected a culture

Human marrow stromal cell line 28s

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Fig. 1. Growth morphology of the human stromal cells. When grown on plastic thecells have a characteristic bipolar shape (A, SEM, X 750) and the cell surface showsnumerous microvilli (B, SEM, x 7500). When the cells achieve contact inhibition ofgrowth (confluence), the morphology becomes epithelial-like (c, living cell, phase-contrast, x 350). After a few weeks of confluence the monolayer is often disorganizedin some areas; over-lapping cells are observed (E, X 200). In such areas, large amountsof extracellular matrix can be observed by phase-contrast or after May-Grunwaldstaining (F, X 600). The cells do not form discrete colonies when cultured in collagengel matrix, but form a typical 3-dimensional network. Most frequently the cells are bi-or tripolar but some multipolar figures are observed (D, X 200).

D


containing a single large colony of fibroblastic cells and a few attached macrophages.The culture was trypsinized and the detached cells were plated without dilution in anew culture flask. Subsequently we found that a 'pure* population of stromal cellscould be obtained after 4 passages; the contaminating macrophages being pro-gressively eliminated by dilution at each passage. One of the cell lines isolated hasnow been maintained for 10 months by serial passage and will be discussed in somedetail.

General characteristics

The morphology of the cells was homogeneous. The cells were fibroblast-likeadherent cells (Fig. 1 A-F), generally bipolar but sometimes tripolar, proliferating toconfluence as a monolayer and becoming contact-inhibited. However, some irregu-larities were observed in the monolayer. Underlapping cells were seen (Fig. IE-F)

200

E 160

80

a 40

Days 10 20 30 45 60 90

P a s s a g e 1 2 3 4 5 6 7 8 9 1 0 1 1

Fig. 2. Kinetics of growth of the human stromal cell line. Effect of hydrocortisone(io~e M) in FCS or HS-supplemented medium. The cell line could be regularly-passaged for months when cultured in medium supplemented with FCS (O-O)-However, in the presence of FCS (A-A) or HS ( • - • ) supplemented with io~' M-HC, the doubling time increased and eventually no further growth occurred.

and a multi-layer pattern was even observed in some areas. When regularly sub-cultured, the cell line did not show any sign of ageing or degeneration. However, ifcultured for several weeks after confluence is reached, the morphology changes andthe cells become epithelial-like (Fig. ic). When these cells were subsequently sub-cultured, they produced multinucleated cells with a very low capacity to proliferate.When subcultured before confluence, the growth was exponential. Routinely, between5 x io2 and io3 cells per cm8 were passaged at each interval and subculture was per-formed when the concentration was about io4 cells per cm2. The doubling time ofthe population was calculated over a period of 3 months of culture (corresponding to12 passages and an estimated number of 65 generations). The doubling time of thepopulation varied between 22 and 42 h (Fig. 2).


Effects of serum and growth promoters

The cell growth was low when medium was supplemented with a concentration of5 % FCS. Between 5 and 10% FCS, the rate of growth progressively increased, theoptimal concentration being between 10 and 15%. Horse serum (HS) at concentra-tions greater than 10% led to arrested growth after one or two subcultures. Similareffects were observed with medium containing FCS and io~8 M-hydrocortisone (HC).Medium containing HS and HC (IO~ 6 M) was inhibitory, (Fig. 2, Table 1). Cellsarrested in growth by HS or HC showed a pattern of ageing similar to that reportedfor long periods of confluence. Growth promoters such as epidermal growth factor

Table 1. Conditions of growth and effects of growth promoters

Conditions of growth^

FCS, s%1 0 %

1 5 %

HS, 10%2 5 %

HS, I O % + I O - ' M - H C

FCS, IO%+IO-«M-HC

FCS, IO % +EGF, ioo ng/mlFGF, io ng/ml

CT, I O - ' M

CT, IO- 1 0 M

Insulin, io/Jg/ml

Carrageenan, io/ig/ml

• The cells were platedwere trypsinized, the cells

First passage

No. ofcells perculture

(io»)

3-86-i

6-8i - 6

0-920 4 8

2 - 1

6 46-32-9

6 77-1

6-5

Averageno. of

doublings

5-255 9 36-09

43-2

2 2 6

439

65-974-86

6 0 7

6 1 5

6-02

at a concentration of io*counted, and

t See Materials and methods.re-plated at

•

Averagedoubling

time

322 8 3276

4 2

52'574'338-3

282 8 1

346

2 7 7

27-328

/flask (25io4/flask

Second passage*A.

No. ofcells perculture

(io6)

2-95"86 2

0-9

o-s0 8

1 8

6 1

6-5

3-1

6-56-8

5-4

ml). After

Averageno. of

doublings

4 85-85 93-1

2-3

3

4- i

5 96

4 96

6-i

5 7

Averagedoubling

time

352 9

2 8 5

54'27 3 1

56

41

28528

3428

27-5

2 9 5

1 week, the cultures

(EGF; 100 ng/ml), fibroblast growth factor (FGF; 10 ng-ml) or cholera toxin(CT; io~10 M) had no effect on cell growth. Horse serum (HS; 10-20%) with hydro-cortisone (io~8 M) or CT (at high concn, io~7 M) were inhibitory. Carrageenan, apotent inhibitor of macrophage and monocytes, had no effect (Table 1).

Growth in soft gels and plastic, and radiosensitivity

The cells did not grow in 0-3 % agar gel, but did proliferate in collagen gel to forma dispersed cell matrix (Fig. ID). When plated on plastic, at 4-5 x io2 cells per cm2,active growth occurred but the cells migrated and rarely formed colonies. When


plated at very low densities (10-50 cells per cm2) no growth occurred, althoughirradiated cell feeders (io2 cells/cm2) could be used to promote cell proliferation. Inthis case, after 4-7 days of culture discrete foci were observed. Although these weredifficult to count, the assay provided a means of testing the radiosensitivity of thestromal cells.

A single cell suspension obtained by trypsinization was submitted to serial doses of137Cs irradiation at 4 °C. The cells were then plated at cell concentrations rangingfrom io2 to io3 cells per 10 ml flask (on irradiated feeder layers) and examined 2weeks later for colony growth. Alternatively, cells were plated at 5 x io3 cells perculture flask (in the absence of irradiated feeders). Before confluence occurred incontrol cultures, the cells were trypsinized and counted.

Similar results were obtained in both assays, giving a D3 7 between 100 and 150 rad

(Fig. 3).

0 100 300 500Irradiation (rad)

Fig. 3. Sensitivity to 1"Cs irradiation. Irradiated cells were plated at concentrationsranging from io1 to i o ' cells per flask, and 2-5 x 10s lethally irradiated cells asfeeders after 2 weeks. In a similar experiment 5 x io3 cells were plated in a 25 cm1

culture flask and cultured until confluency occur in the control bottle. Cells weretrypsinized and counted.

Phagocytic ability

Tests for phagocytosis were carried out on the cells after the fourth passage andafter 6 months of serial sub-culture. We used the 3 different techniques described inMaterial and methods (opsonized yeast, glutaraldehyde-fixed SRBC, and latexparticles). A primary culture (not subcultured) was also tested. It was found thatprimary cultures show variable numbers of phagocytic cells, but the stromal cell linewas negative after both 4 passages and 6 months of serial passages.


Histochemistry

The results are summarized in Table 2. The cells were negative for membraneAlk-Pase activity but the cytoplasm was stained strongly for Ac-Pase, mainly in theperinuclear region. The acid phosphatase reaction was not diffuse but restricted toclusters of heavily stained granules (Fig. 4 A, B). The pattern of staining is character-istic of a strong Ac-Pase lysosomal reaction.

Table 2. Cytoplasmic and membrane markers: comparative studies of thestromal cell Une with primary cultures

Human stromalMembrane and cytoplasm markers cell line

Positive cells in theprimary culture

a-Naphthyl acetate esteraseNaphthol ASD chloroacetateNaphthol ASD acetate esterase

(non-specific esterase)

Alkaline phosphatase (Alk-Pase)Acid phosphatase (Ac-Pase)

Fc membrane receptor(Agg. Hu IgG receptor)

Factor VIII antigenDirect immunofluorescenceSandwich immunofluorescence

Collagen synthesisReticulinPeriodic acid-Schiff

Monocytes, macrophages ( + +)Granulocytes (+ +)Monocytes (+)Macrophages (+)Fibroblast-like cells ( ± )

N.D.Monocytes (±)Macrophages (+ +)Mononuclear leucocytes (+ )*

Human endothelial cells ( + + + )t

Unidentified adherent cells (+ + )

N.D.

• Adherent population from human blood leucocytes (methocel sedimentation),t Primary culture of human endothelial cells from umbilical cord.N.D., not determined.

Thin sections by TEM clearly showed large numbers of both primary and secondarylysosomes within the cytoplasm (Fig. 5). The cells were negative for chloroacetateesterase, but clearly positive for naphthyl esterase (Fig. 4D). Appropriate controlswere done on primary cultures.

Membrane receptors for binding aggregated human immunoglobulin G (IgG) wereinvestigated using 2 concentrations of IgG (0-2 mg/ml, 1 mg/ml). The highest con-centration was reported to be sensitive enough to detect low concentration or lowactivity receptors on non-lymphoid cell membrane (Papamichail et al. 1979). All thecells were negative. Peripheral-blood adherent leukocytes were used as a positivecontrol. The presence of factor VIII antigen was investigated by the direct andindirect immunofluorescence techniques. The tests were negative in both cases.

Collagen synthesis was demonstrated by silver impregnation, a specific histologicajstaining method (Pearse, i960). Before confluence, individual cells were labelled witha thin black membraneous deposit characteristic of reticulin (Fig. 4F). In confluent


X'

Fig. 4. Histochemical analysis. Stromal cell fnonolayer stained for acid phosphatase(A, x 100). The activity can be detected in all cells and is concentrated in the peri-nuclear region. The cytoplasmic extensions of celk are weakly positive or negative(B, x 400). For comparative purposes, a population of stromal cells derived from boneendosteum was cultured for 3 weeks in collagen gel matrix. The bone trabecule wasremoved by dissection before staining for Ac-Pase (c). These cells show a uniformstrong Ac-Pase activity. D. The diffuse non-specific esterase reaction in the cytoplasmof the human stromal cells, E. The deposit of extracellular materials on plastic bygrowing stromal cells (x 7500). F. The thin membranous labelling of reticulin bysilver impregnation (Pearse, i960).


Fig. 5. Vertical sections through stromal cell cultured on a plastic substratum andembedded in situ. A. Cytoplasmic region showing profiles of endoplasmic reticulumand numerous primary lysosomes (/)• Extracellular matrix is also apparent, x 23000.B. Cytoplasmic region of a different cell showing numerous secondary lysosomes (*)and residual bodies. A dense aggregation of sub-plasmalemmal microfilaments isalso apparent (arrows), x 22000.


cultures, deeply stained bundles of extracellular collagen fibres were apparent. Afibrous extracellular matrix can be shown on the cell layer (Fig. IF) as well as adeposit between the cell and the plastic surface (Figs. 4E, 5 A).

Induction of adipocyte differentiation by hydrocortisone

Cells were cultured in Fischers' medium with FCS and HC (io~6 M) or HS andio~6 M-HC. The cells were maintained at confluence and fed weekly. Thirty daysafter confluence occurred the first adipocytes were observed in cultures containing

Fig. 6. Adipocyte conversion of stromal cells. A. Cluster of cells at various stages ofadipocyte differentiation, x 750. B. Early adipocyte with small fat vesicles, x 3000.c. Fully mature adipocyte. x 10 000.

HC in the culture medium. Small refractile lipid vacuoles accumulating in the peri-nuclear region were characteristic of a preadipocyte stage. Eventually, the fat vesiclesenlarged, first in the proximity of the nuclei, then progressively in peripheral exten-sions of the cell cytoplasm. The cell, originally very flat, increased in volume androunded up when giant fatty vacuoles developed (Fig. 6B, C). Finally the cells in mostcases retracted from the plastic.

It is of some interest that transformation to adipocytes is not synchronous but has


a tendency to occur at foci (Fig. 6 A). The cells do not seem to be uniform in theircapacity to form large round adipocytes; some cells remain for very long periods inculture, with the appearance of preadipocytes (small lipid vacuole9). We have alwaysfound that the induction of adipocyte conversion needs arrested growth (by con-fluence). It is of some interest, however, that the subculture of a confluent layer, after2 weeks growth in the presence of HC, does not prevent the formation of fat cells.

The kinetics of ["Cjpalmitic acid incorporation into fat was investigated beforeand after adipose induction. We observed a faster uptake of the label in the fat cellculture (Fig. 7). However, after the lipids were extracted, the distribution of the labelin lipid and non-lipid material in the induced and non-induced cells was found to besimilar. Therefore, although both induced and non-induced cells take up the label,the rate of uptake rather than the distribution is the distinguishing feature.

100 -

01 2

Time (h)

Fig. 7. Rate of [14C]palmitic acid uptake by stromal cells before ( • - # ) and after(A-A) adipocyte conversion.

Other cell lines

Apart from the cell line discussed above, we have isolated and maintained 3 othercell lines with similar if not identical characteristics to those described above.

DISCUSSION

In this study, we have isolated several human bone-marrow stromal cell lines. Oneof these lines has now been maintained in culture for 10 months and has a fibroblasticmorphology on plastic and in collagen gels. It grows as a monolayer and achievescontact inhibition but does not clone in soft agar. It synthesizes retictilin and thenlarge amounts of collagen after confluence is reached. The cells do not express factorVIII antigen but are positive for non-specific esterase and strongly positive for

294 M- Lanotte, T. D. Allen and T. M. Dexter

Ac-Pase. However, the cells are not phagocytic, do not express Fc receptors (i.e.aggregated human IgG receptors) and are Alk-Pase negative. The cells can beinduced to differentiate to adipocytes by addition of hydrocortisone.

The characteristics listed above make it difficult to categorize the cells into anyknown cell lineage. Consequently, we will discuss each set of data separately.

(a) The cell line was derived from a fibroblastic colony (CFU-F) grown in primarybone-marrow stromal culture. In many of its features it may be classified as a fibro-blast-like cell: in its morphology, growth pattern, active biosynthesis of collagen.However, we have observed a very high Ac-Pase activity, which seems to be localizedin lysosomes. In addition, the cell line is also clearly a-naphthol esterase positive.These data may indicate that the cell line belongs to the monocyte-macrophagelineage. However, the cells are not phagocytic and do not express Fc receptor. Apartfrom the fact that the cells are Ac-Pase positive, our cell line has all the characteristicsof the CFU-F progeny observed (Castro-Malaspina et al. 1980) in primary cultureof human marrow.

It has been reported recently (Westen & Bainton, 1979) that phosphatase activitycan be used to classify the murine stromal elements into 2 different types: (1) afibroblast-type of reticulum cell, concentrated near the endosteum and characterizedby having the Alk-Pase activity associated with the plasma membrane; (2) a macro-phage-type of reticulum cell with lysosomal acid phosphatase activity, which isuniformly distributed throughout the marrow. Unfortunately, our cell line does notfit into this simple classification. It might be classified as a fibroblast-type reticulumcell but is clearly Ac-Pase positive. Alternatively, it might be a macrophage-like cellbut is not phagocytic and does not express Fc receptor. In this respect, it is significantthat when endosteum-associated stromal cells were cultured in a 3-dimensionalcollagen gel matrix (Fig. 4c) the stroma cells proliferated. These cells, also having afibroblastoid morphology, were uniformly Ac-Pase positive and were not phagocytic.

It is possible that our cell line is similar to the cell types described first by Frieden-stein (Friedenstein et al. 1974, 1976) and other authors (Blackburn & Goldman, 1980;Toogood et al. 1980; Castro-Malaspina et al. 1980; Wilson, O'Grady, McNeil &Muhum, 1974; Wilson et al. 1978), which are thought to be mesenchymal elements.These cells are supposed to be multipotent; apart from having a self-perpetuatingcapacity, they are able to produce bone in vivo, and form a haemopoietic inductivestroma.

The pertinent features in the cell line we obtained are: firstly the Ac-Pase activity,and secondly the striking observation of bundles of extracellular collagen. We ob-served that the cells contained rough ergastoplasmic reticulum and also regions richin primary lysosomes (in some places the cytoplasm of these cells was also filled withnumerous secondary lysosomes) (Fig. 5). This seems to indicate that the cells simul-taneously express 2 activities, which are usually mutually exclusive: biosynthetic anddegradative activity. Such situations (however uncommon) have been reportedpreviously (Napolitano, 1964; Ten Cate, 1972; Deporter & Ten Cate, 1973). It hasalso been reported that chondrocytes, in the preliminary stage of calcification of theirmatrix, express Ac-Pase in association with membrane vesicules when they are actively


involved in glycosaminoglycan synthesis (Hall, 1978). In the case of the humanstromal cell line reported above, the strong Ac-Pase activity observed may be ex-plained as a physiological response to an excessive synthesis of extracellular matrix inrelation to the condition of growth in culture. It will be interesting to determine if thisswitch is reversible.

If the above argument is accepted, it follows that all Ac-Pase cells detected in thebone marrow must not be assigned definitely to a macrophage-type reticulum cell.A pertinent observation (Westen & Bainton, 1979) was that the Ac-Pase stromalcells are associated with extracellular reticulin; and secondly, although a rare occur-rence, such cells are capable of mitosis.

(b) The human stromal cell lines do not have the characteristic morphology ofendothelial cells. Furthermore, we did not find Weibel-Palade bodies and the cellswere negative for factor VIII antigen. However, even if they are not derived fromthe endothelial components of the sinus, it still remains difficult to exclude all relation-ship with the pericyte-like elements of the marrow.

(c) The cells undergo a conversion to adipocytes when treated with hydrocortisone.The morphological features of these adipocytes are very similar to those observed inprimary culture and in long-term bone marrow cultures (Allen & Dexter, 1976 a, b;Dexter et al. 1977; Toogood et al. 1980; Gartner & Kaplan, 1980). The occurrenceof fat cells is a well-documented observation in bone marrow in vivo as well as inlong-term cultures, although as yet there is no agreement about the origin of the fatcells and their role in the inductive environment. In long-term marrow cultures theadipocyte conversion seems to depend upon corticosteroid hormones, since hydro-cortisone can be used successfully to supplement the adipocyte-promoting propertiesof many batches of serum. We have also observed that murine fibroblastic stromal-cellcolonies (unpublished results), growing in collagen gel, can be induced to differentiateinto fat cells after 3 weeks incubation with hydrocortisone. It is of some interest thatinsulin (shown to facilitate adipocyte conversion in other systems; Green & Kehinde,1975) has no such effect upon marrow stromal cells (Greenberger, 1978; Lanotte,unpublished observations).

In this respect, it may be relevant that monocytes from human peripheral bloodspontaneously differentiate into adipocytes in soft agar gels (Zucker-Franklin, Grusky& Marcus, 1978). The precursor cells were phagocytic and possessed lysosomes andFc receptors. It has also been demonstrated by electron microscopic studies that bonemarrow pericytes from sinus can differentiate into fat cells (Iyama, Ohzono & Usuku,1979). Consequently, the fact that our cell line can be induced to differentiate intoadipocytes does not help to assign it a place in the mesenchyme or haemopoieticlineage. However, such a cell line may serve to facilitate the study of adipose conver-sion in the marrow and investigations of defects in marrow adipogenesis. It will servealso as a tool for understanding the processes of the replacement of medullary haemo-poietic tissue by fat tissue in physiological as well as pathological conditions.

Our results, with primary collagen cultures and the cell lines described here,indicate that caution must be exercised in assigning a cell to a particular cell lineagebased upon limited aspects of morphology or cytochemistry. In the cell lines reported,


a multi-parameter analysis has demonstrated the difficulty in classifying the cells asbelonging to the fibroblast, endothelial, adipocyte or monocyte/macrophage path-ways. Because of *he lack of similar information on other 'fibroblast' cell lines studiedfollowing culture of marrow cells, we cannot make comparisons with our data.However, at present we are attempting to define the growth conditions necessary forthe development of other marrow stromal elements, e.g. fibroblasts and endothelialcells. In addition, we are attempting to subclone the cell type reported here in orderto investigate heterogeneity in the phenotype.

Finally, the availability of such a human bone-marrow stromal cell line will beuseful for studies in vitro on radiation damage to the marrow environment. Hopefully,as an element of this environment, it will be useful for the construction in vitro of afunctional human haemopoietic inductive microenvironment and the studies ofcellular interactions or regulators produced therein. In a following paper, the role ofthis human stromal cell line in granulopoiesis in vitro, and its synergestic effects oncolony-stimulating factors, will be reported.

The authors wish to thank Dr Williams (Pathology Department) who helped in the histo-chemical work and Drs S. Kumar, A. Schor (Medical Oncology Department) and J. Garlandfor their assistance in the anti-factor VIII test and Agg. Hu IgG binding assay.

This work was supported by the Medical Research Council and Cancer Research Campaign.T.M.D. is a fellow of the Cancer Research Campaign. M.L. is a visiting fellow supported bythe CNRS (France) and the Royal Society (U.K.).

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{Received 8 December 1980)

HISTOCHEMICAL AND ULTRASTRUCTURAL ...HISTOCHEMICAL AND ULTRASTRUCTURAL CHARACTERISTICS OF A CELL...

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