Proc. NatL Acad. Sci. USAVol. 80, pp. 4847-4850, August 1983Medical Sciences

Phenomenon of formation of giant fat-containing cells in humanbone marrow cultures induced by human serum factor: Normaland leukemic patterns

(hematopoietic microenvironment/leukemias/hormones/hemopoietic precursor cells)

INNA A. SVET MOLDAVSKAYA*, SVETLANA N. ZINZAR*, GEORGE J. SVET MOLDAVSKY*, ZALMEN ARLINt,CAROL VERGARA*, BENJAMIN KOZINERt, BAYARD D. CLARKSONt, AND JAMES F. HOLLAND**Department of Neoplastic Diseases, Mount Sinai Medical Center, New York, New York 10029; and tHematology-Lymphoma Service, Department of Medicine,Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Communicated by Robert A. Good, January 24, 1983

ABSTRACT Normal human sera induce the formation of fat-containing cells (FCC) in human bone marrow cultures. A nearlycomplete monolayer of FCC is formed after 7-14 days of culti-vation with 20% human sera in the medium. FCC-inducing ac-tivity (FCCIA) is nondialyzable through 14,000-dalton cutoffmembrane and is stable at 560C for 30 min. Abundant FCCIA wasfound in 83% of normal human sera but in only 20% of sera fromuntreated patients with different hemopoietic disorders and in 32%of treated leukemic patients. It is suggested that FCCIA may beinvolved in regulation of the bone marrow microenvironment andthat it varies in normal individuals and in patients with differentdiseases.

The important role of stromal cells in the proliferation and dif-ferentiation of hemopoietic cells has been shown both in vivo(1-4) and in vitro (5-16). In the continuous bone marrow cul-ture system, proliferation of hemopoietic cells is believed todepend upon the development of a monolayer of adherent cellscomposed of fibroblasts, macrophages, epithelial cell-like cells,and fat cells (or adipocytes). Hydrocortisone and horse serumat high concentration or a mixture of fetal bovine and horse serumare required for the development of all of these cells. Someresearchers have especially emphasized the role of fat cells inhemopoiesis (6, 8, 11).

In an attempt to increase the number of fat cells in humanbone marrow cultures, we substituted human serum for xen-ogeneic sera. An almost pure population of giant fat-containingcells (FCC), not adipocytes, was formed in human bone marrowcultures containing 20% human serum. In the present study,the distribution of FCC-inducing activity among normal andpathological human sera was investigated.

MATElMAL AND METHODSPatients and Healthy Donors. Patients with different types

of acute and chronic leukemias were studied before treatment,during relapse (before new chemotherapy had begun), duringchemotherapy, and in remission. Leukemias were diagnosed onthe basis of cellular morphology, cytochemical stainings, de-termination of terminal deoxynucleotidyltransferase, and studyof immunological markers.

Blood and Bone Marrow Specimens. Blood from leukemicpatients and healthy donors was collected individually in evac-uated tubes. After clotting the blood was stored overnight at4°C and then centrifuged at 750 x g. The supernatant (clearserum) was then collected, stored at -70°C, defrosted, heated

at 560C for 30 min, and used in experiments.Bone marrow was obtained from normal donors and from

patients by means of puncture of the posterior iliac crest. Bonemarrow cells were aspirated in sterile syringes that containedpreservative-free heparin solution in Dulbecco's phosphate-buffered saline to a final concentration of 20 units per ml ofmarrow aspirate. Cells were centrifuged at 750 X g for 10 min.Buffy coats were collected and washed twice in RPMI 1640 me-dium. All specimens were collected from informed voluntarydonors in conformity with institutional and federal guidelines.

Cell Cultures. After two washings with RPMI 1640 medium,the bone marrow buffy coat cells were placed into 75- or 25-cm2plastic flasks (Falcon) or in 35-mm Petri dishes with grids (Lux)in concentrations of 1-2 X 106 cells per ml in RPMI 1640 me-dium supplemented with 20% fetal bovine serum (GIBCO) or20% human serum with penicillin and streptomycin. For mor-phological study, coverslips were placed in the dishes. The cul-tures were incubated at 370C in a humidified atmosphere with5% CO2.

Staining. Bone marrow smears and coverslip cultures werestained with Giemsa-Romanovsky stain after May-Grunwaldfixation. For determination of the presence of lipids, FCC cul-tures were fixed with 10%o neutral formalin and stained by oilred 0 or Sudan black B (17).

Study of Cultures and Cell Count. On days 7-8 and 14-15,FCC were counted in 7-10 2-mm2 squares located in differentparts of the dish and the counts were averaged. The mean num-ber of FCC per square was graded according to the followingsystem: >300 per square = 4+; 100-300 = 3+; 50-100 = 2+;5-50 = 1+; <5 =+; and none =- 0.

Standard Human Serum. Serum from two healthy donorswas used as a standard of FCC-inducing activity (FCCIA) ineach experiment, along with other sera. Each result from thestudy of sera of patients with hematological diseases was com-pared with results from the standard sera. Furthermore, resultsfrom leukemia sera were taken into consideration only whenthey were checked against normal bone marrow cells that re-sponded to standard serum with 3+ or 4+.

RESULTSWhen human normal bone marrow cells (49 cases) and chronicmyeloid leukemia (CML) bone marrow cells (70 cases) werecultivated with 20% fetal bovine serum, fibroblasts appeared atthe second week of cultivation and developed into a monolayer

Abbreviations: CML, chronic myeloid leukemia; FCC, fat-containingcells; FCCIA, FCC-inducing activity; RAEB, refractory anemia withexcess blasts.

4847

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4848 Medical Sciences: Svet-Moldavskaya etalP

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FIG. 1. Human serum-induced FCC formation in normal bone marrow. (A and B) Monolayers of FCC in 8-day culture. (A, x 25 B. x 41.) (C)Conglomerate surroundedby round cells in l8-day culture. (X41.) (D-F) Fourteen-day culture. (D) Huge conglomerate (x41); (E) huge conglomeratereleasing FCC into medium (x41); (F) single'FCC and groups of FCC surrounded by monolayers of round cells (x 25).

after 3-4 weeks. Only a few FCC were found in some culturesof normal bone marrow with fetal bovine serum, and they rap-.idly disappeared. When human normal bone marrow cells werecultivated with 20% human serum, from healthy donors, be-ginning 72-96 hr after plating all cultures showed single ovoid

or polygonal cells 80-300 jxm in diameter with abundant darkcytoplasm and one often large central or eccentric nucleus.. Thesecells were stained strongly for lipids by oil red 0 and Sudanblack and were designated as FCC. The number of FCC in-creased gradually from 2-10 to more than 300 cells per square.

Proc. Natl. Acad. Sci. USA 80 (1983)

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Medical Sciences: Svet-Moldavskaya et al.

Table 1. FCC formation in normal bone marrow cell culturesinduced by sera from healthy donors

Serum donor FCC formationAge, 7-8 14-15

No. Sex yr days days1 M 192 F 203 F 214 F 215 F 236 F 247 F 258 F 279 F 2810 F 2811 M 2812 F 2813 F 2914 F 3015 M 3016 F 3117 M 3218 F 3219 F 3320 F 3321 M 3422 M 3623 F 3624 M 3625 M 3726 F 4027 F 4028 F 4429 M 91

+ 1++ 2++ 1++ 3+1+ 3+1+ 2++ 3+1+ 1+1+ 4++ 3+1+ 4++ 2+2+ 3+2+ 3+2+ -3+0 1+3+ 3+1+ 1+1+ 2+1+ 3+0 4+1+ 3++ 3+1+ 2+1+ 3+2+ 3+1+ 4+2+ 4+2+ 4+

Proc. Nati. Acad. Sci. USA 80 (1983) 4849

Table 2. FCC formation in normal bone marrow cell culturesinduced by sera from patients with hemopoietic disorders

Serum donor FCC formationAge, 6-7 14-15

No. Sex yr Diagnosis* Phaset days days1 M 21 ALL A 0 +2 M 18 ALL A + +3 M 17 ALL T cell A 1+ 1+4 M 17 ALL T cell RM 0 +5 F 40 AML A 1+ 2+6 M 45 AMOLV RPS 0 1+7 M 75 AMMOLV RPS 0 1+8 M 25 Biphenotypic RM 3+ 2+9 M 48 Hairy cell A 0 010 M 34 Hairy cellt Pancyt. 3+ 4+11 F ? AA - 0 1+12 F 26 AA - 2+ 1+13 F 47 RAEBt - + +14 F 54 RAEBt - 0 t15 M 51 RAEBt - + +16 M 31 RAEBt - 1+ ±17 M 68 Preleukemia - 0 ±18 F 50 CLL Chr 0 1+19 F 64 CML-Bl A 1+ 1+20 F 46 CML-Bl A 1+ 2+

?, Data lost or unknown.* ALL, acute lymphoid leukemia; AML, acute myeloid leukemia; AMOL,acute monocytic leukemia; AMMOL, acute myelomonocytic leuke-mia; biphenotypic, myeloid plus lymphoid leukemia; AA, aplasticanemia; RAEB, refractory anemia with excess (approximately 30%)blasts, formerly known as "smoldering" leukemia; CLL, chronic lym-phoid leukemia; CML-Bl, CML blastic crisis.tA, acute; RM, remission; RPS, relapse; Pancyt., pancytopenia; Chr,chronic.

tPatients received cis-retinoic acid.

During 7-14 days of cultivation, the bottoms of the flasks anddishes became covered with monolayers of almost pure giantFCC (Fig. 1 A and B). FCC were strongly adherent but couldbe detached by 0.5% trypsin with EDTA and replated. After2-3 weeks of cultivation, a special kind of conglomerate of cellsappeared (Fig. 1 C and D). In each conglomerate the cells werecovered with a dark semitransparent envelope, which disap-peared after collagenase treatment. Some of these conglom-erates remained attached and some became detached and floatedfree, sometimes releasing FCC into the medium (Fig. 1E).Conglomerates and FCC were often surrounded by round, pu-tatively primitive hemopoietic cells (Fig. IF).Among 29 human sera from healthy donors studied, 24 (83%)

induced the formation of many FCC (2+ to 4+; Table 1). FCCIAin sera was not dialyzable through membranes with 6,500- or14,000-dalton cutoff weight. FCCIA was stable after the hu-man sera were heated at 56°C for 30 min.

Study of FCCIA from patients with acute and chronic leu-kemias and other hematological diseases is presented in Tables2 and 3. Only indicator normal bone marrow cells that re-sponded to the standard human serum FCCIA by 3+ or 4+FCC formation were used. Sixteen of 20 (80%) untreated pa-tients (including RAEB patients treated with cis-retinoic acidonly) induced little or no FCC formation (0, ±, or 1+) (Table2).

Thirteen of 19 (68%) treated leukemic patients also exhibitedlow FCCIA (0, ±, 1+) (Table 3). Of special interest are samples12 and 12a. When the patient was in CML chronic status hisserum (sample 12) induced 3+ FCC. After blastic transfor-mation his serum (sample 12a) lost FCCIA (0, activity).

DISCUSSIONThe experiments presented above show that normal human seracontain a factor that induces FCC formation in human bonemarrow. Approximately 83% of sera of healthy donors exhib-ited intensive FCCIA (Table 1). The level of FCCIA is greatlyreduced in patients with leukemia, RAEB, and aplastic anemia.Of these, 80% of untreated patients and 68% of treated pa-tients (Tables 2 and 3) showed very little or no FCCIA. Sera ofpatients with chronic leukemia possessed higher levels of FCCIAthan sera of patients with acute leukemia. In one case (Table3, nos. 12 and 12a), serum taken from a patient in the chronicstage of CML showed a high level of FCCIA; when transfor-mation to blastic crisis occurred, FCCIA was reduced to 0 level.All four cases of RAEB studied showed absence of FCCIA.

Preliminary data based on monoclonal antibody assay iden-tified FCC as macrophages. FCC observed by many others inbone marrow cultivated with xenogeneic sera (horse serum ora mixture of fetal bovine serum and horse serum) appear to beadipocytes on a morphological basis (6, 8, 11).

The role of FCCIA in the pathogenesis of leukemia and otherhematological diseases is uncertain, but it could be related toregulation of the bone marrow microenvironment. Furtherstudies are indicated to determine which cells or organs are re-sponsible for FCCIA, to determine its biochemical composi-tion, and to elucidate its physiological role in normal and ab-normal hemopoiesis.

This work was supported by a grant from the Cancer Research In-stitute, Inc., a grant from the T. J. Martell Foundation for LeukemiaResearch, and National Cancer Institute Cancer Center Grant 5Pll-CA15936.

4850 Medical Sciences: Svet-Moldavskaya et al.

Table 3. FCC formation in normal bone marrow cell cultures induced by sera from treated leukemic patients

Serum donor FCC formation

Age, 6-7 14-15No. Sex yr Diagnosis* Phaset Treatment$ days days

1 M 21 ALL A AAT 2+ 2+2 F 29 ALL RM VCR + P 1+ 1+3 M ? AML A ? 1+ 1+4 F 69 AML A 6-MP 1+ 1+5 M 72 AMMOL RM VCR + P 1+ 2+6 M 51 AMMOL RM AAT + +7 F 31 AMMOL A HU 1+ 1+8 F 20 APL RM HU 3+ 4+9 F 55 CLL Chr Cytoxan + P 0 1+10 M ? CML Chr Myleran 0 1+11 M 60 CML Chr HU + 2+12 M 49 CML Chr HU 2+ 3+12a M 49 CML-Bl (megakaryocytic) A HU 0 013 M 41 CML-B1 A ? 2+ 2+14 M 27 CML (20% blasts in blood) - HU 1+ 1+15 M 24 CML-B1 (lymphoblastic) A VCR + P 0 1+16 M 53 CML-BI (lymphoblastic) A VCR + P 0 1+17 M 53 CML-B1 (lymphoblastic) A HU 0 1+18 M 38 Blastic (unclassified) A VCR + HU 0 1+

?, Data lost or unknown.* Abbreviations as in Table 2; APL, acute promyelocytic leukemia.tAbbreviations as in Table 2.tAAT, 4'-(9-acridinylamino)methanesulfon-m-anisidine + cytosine avincristine; P, prednisone; 6-MP, mercaptopurine.

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Proc. Natl. Acad. Sci. USA 80 (1983)