Leukemia Research Vol. 14, No. 7, pp. 611-616, 1990. Printed in Great Britain.
0145-2126/90 $3.00 + ,00 Pergamon Press plc
D I F F E R E N T I A T I O N INDUCTION OF HL60 CELLS IN A LONG TERM BONE M A R R O W CULTURE OF ACUTE MYELOID LEUKEMIA G. J. OSSENKOPPELE,I. DENKERS, P. WIJERMANS, P. C. HUIJGENS, J. J. P. NAUTA,* R. J. H. BEELEN and M. M. A. C. LANGENHUIJSEN *Department of Haematology and Medical Statistics and Free University Hospital, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands
(Received 17 November 1989. Accepted 20 February 1990) Abstraet--Bone marrow stromal cells are critical for the proliferation and differentiation of hemopoietic stem cells. The hemopoietic microenvironment is reproduced in long term bone marrow culture (LTBMC). Normal LTBMC versus leukemic LTBMC and their stroma conditioned medium were compared with respect to their proliferative and differentiation-inducing capacities. Myeloid leukemic cells (HL60) were layered onto LTBMCs derived from normal volunteers and patients with AML. Differentiation was measured with a comprehensive panel of maturation parameters, i.e. morphology, cytochemistry, quantitative enzyme determination, NBT test and immunophenotyping. Inhibition of proliferation occurred in all cocultures. Clear maturation in monocytic direction was obvious in one culture of HL60 cells layered onto a leukemic stroma. As stroma-derived conditioned medium has no effect, a cellular interaction seems involved. These observations support not only the concept that normal stroma influences leukemic cell growth but also that leukemic stroma can modulate cell growth and maturation.
Key words: Differentiation, HL60 cells, long term bone marrow culture AML.
INTRODUCTION
kemia and from normal volunteers on leukemic cells with respect to proliferation and differentiation inducing capacities.
THE HEMOPOIETIC microenvironment plays a critical role in proliferation and differentiation of stem cells. Dexter et al. [1] developed a system for the long term culture of murine m a r r o w , which has been adapted to studies of h u m a n m a r r o w [2, 3, 4]. These long term bone m a r r o w cultures ( L T B M C ) are characterized by a stromal layer c o m p o s e d of fibroblasts, macrophages, fat cells and endothelial cells producing an extracellular matrix possibly responsible for microenvironment specificity [5, 6]. L T B M C has allowed the study of the h a e m o p o i e t i c microenvironment providing a model for the complex interactions between stroma cells, extracellular matrix and haemopoietic stem cells from normals as well as from patients with hematological disorders. We recently introduced a comprehensive panel of quantitative and qualitative maturation p a r a m e t e r s useful in the investigation of myeloid differentiation in cell lines and freshly obtained leukemic cells [7, 8]. In this study we examined the influence of stromal cells derived from patients with acute myeloid leu-
MATERIALS AND METHODS
Long term bone marrow culture (LTBMC) Bone marrow cells were obtained from two normal volunteers and three consecutive patients with acute myeloid leukemia (clinical data are summarized in Table 1). Mononuclear cells were isolated after centrifuging on Ficoll-Hypaque density gradient (d = 1.077) at 400g for 30 min and washed twice in Hank's-BSA 0.1%. Bone marrow mononuclear cells were suspended in LTBMC growth medium (alpha-MEM supplemented with 400 mg/liter glutamine, 40 mg/liter inositol, 10 mg/liter folic acid, 12.5% horse serum, 12.5% fetal calf serum, 10-6M hydrocortisone and antibiotics) to give a final concentration of 1 × 106/ml; 5 ml aliquots were added to 25 cm2 culture flasks (Falcon). The cultures were incubated at 37°C in humidified 5% CO2 and fed weekly by complete replacement of the LTBMC growth medium until the bottoms were covered by confluent layers of stromal cells and hemopoietic progenitors had disappeared. Stroma conditioned medium (SCM) Supernatants discarded before the LTBMCs were used for coculture experiments were collected and added to HL60 cells as described below.
Correspondence to: G. J. Ossenkoppele, Free University Hospital, Department of Haematology (BR238), De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. 611
612
G . J . OSSENKOPPELE et al.
Cell line The HL60 promyelocytic leukemia cell line was maintained in suspension culture in alpha-MEM (Gibco, Grand Island, New York) containing 20% fetal calf serum (FCS) (Flow Lab, Rockville, MD), heat inactivated at 56°C for 30 min, with penicillin and streptomycin. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in air. Coculture o f HL60 cells onto L T B M C and with SCM HL60 cells were seeded at a density of 1 × 106ce11/ ml onto LTBMC. After 96 h HL60 cells were harvested, attached cells were detached by gentle pipetting with PBS. Also HL60 cells were added to SCM at a density of 1 × 106 cell/ml and harvested after 4 days. HL60 control cells grew in LTBMC growth medium for 4 days. HL60 control cells grew in LTBMC growth medium for 4 days. Vitality of harvested cells was always >90% as determined by trypan blue dye exclusion. Cell counts Cell number was determined by flow cytometric technique on an AI Cell Counter 134 (Analysis Instruments). Cytochemistry After 96 h in culture cells were collected on slides using a Shandon cytocentrifuge. Cytospin slide preparations were stained according to May-Grunwald-Giemsa for assessment of morphology. The cells were also stained with Sudan black and alpha naphthyl acetate esterase stains as previously described [19]. Differential counting of at least 100 cells was performed by two members of our study group independently. In each cytochemical stain 100 cells were judged for positivity by at least two of the investigators. Intracellular enzyme activity Cells were lysed by freezing them three times in liquid nitrogen. Cell fragments were spun down at 7000g for 4 minutes, the collected supernatant was used for enzyme determination. Techniques included minor variations, as described in detail elsewhere [14], of standard methods for the estimation of myeloperoxidase [10], acid esterase using both alpha naphthyl acetate and alpha naphthyl butyrate as a substrate [11]. The amount of enzyme is expressed as units per 106 cells and as units per mg protein. The amount of protein in the cell lysate was determined according to the method of Lowry et al. [12]. Nitro blue tetrazolium ( N B T ) reduction The reduction of NBT was measured quantitatively. One hundred microliters of cells (0.5 x 106/ml) in tr-MEM containing 20% FCS were incubated at 37°C for 15 min with 100 ~tl NBT (0.5 mg/ml) and 2 ~tl 10 ~tM freshly diluted 12-O-tetradecanoyl phorbol 13-acetate (TPA). A native preparation was made in which the percent of cells containing blue black deposits of formazan was determined by evaluating 200 cells. Cell diameter Cell diameters were measured in cell suspensions by means of an Elzone 80XY Volume Counter (Particle, Data, U.S.A.), which measures and plots cell diameters according to the coulter principle in 128 channels.
TABLE 1. CLINICALDATA OF THREE PATIENTSWITH AML Patient Sex Age 1 2 3
f f m
81 30 52
FAB Cytogenetics M5b M4 M4
46 XX 46 XX (inv9) 46 XY
% Blasts
CR
92% 80% 89%
+ +
Immunophenotyping Cell membrane phenotypes were determined by using commercially available monoclonal antibodies combined with an immunoperoxidase technique. Antibodies included: HLA-Dr (Dakopatts, Denmark), E B M l l (Dakopatts, Denmark) [13] a monocyte-macropbage specific antibody and RFD7 (kindly provided by Dr L. W. Poulter) [14] a marker for mature macrophages. Statistical evaluation Data analysis was performed using Student's t-test. When the response variable was a proportion, the statistical analysis was not performed on the values themselves but on the values transformed by the angular transformation, A = arcsin(X/p). When proportions are close to either 0 (zero) or 100 the normality assumption applies better to these transformed values. The resulting confidence limits were transformed back to the original scale.
RESULTS The proliferation of H L 6 0 cells layered o n t o the normal L T B M C s as well as o n t o the leukemic L T B M C s was inhibited. A l s o g r o w t h in the presence of SCM of either origin was diminished. T h e s t r o m a conditioned media derived f r o m all L T B M C did not induce any significant change in the m e a s u r e d differentiation parameters. A f t e r coculture with normal L T B M C , no i m p o r t a n t m o r p h o l o g i c a l change was o b s e r v e d in H L 6 0 cells. N e i t h e r did quantitative e n z y m e d e t e r m i n a t i o n reveal a significant change in c o n t e n t of protein, myeloperoxidase or acid esterase. N o c h a n g e in antigenic determinants was measured. H o w e v e r , in the H L 6 0 cells cocultured with the leukemic s t r o m a layer of patient two, clear signs of m a t u r a t i o n were o b s e r v e d concurrent with inhibition of cell growth. As is shown in Figs l a and l b a decrease in nuclearcytoplasmic ratio, loss of nucleoli and d e v e l o p m e n t of vacuoli was exhibited pointing to m a t u r a t i o n in monocytic direction. T h e p e r c e n t a g e of Sudan black positive cells r e m a i n e d 100% t h o u g h intensity decreased. T h e strong positivity for non specific acid esterase was most striking (Figs 2a and 2b). These m o r p h o l o g i c findings are confirmed by a decrease in m y e l o p e r o x i d a s e content and an increment of acid esterase a m o u n t by using alpha naphthyl acetate as well as alpha n a p h t h y l butyrate as substrate. Antigenic d e t e r m i n a n t s were compatible
FIG. la. HL60 control cells after 4 days culture in medium (MGG-staining).
FIG. lb. HL60 cells after coculture onto long term bone marrow culture from patient two (MGG-staining). Monocytic differentiation is shown. 613
FIG. 2a. HL60 control cells after 4 days culture in medium (non specific esterase staining).
FIG. 2b. HL60 cells after coculture onto long term bone marrow from patient two. This non specific esterase staining is clearly positive. 614
Differentiation in a long term bone marrow culture
615
with a more mature celltype as shown by a moderate increase in H L A - D r positive cells and a remarkable increase in E M B l l positive cells. Coculturing HL60 cells with stroma of patients one and three did not lead to maturation.
< ©
DISCUSSION Z
0145-2126/90 $3.00 + ,00 Pergamon Press plc
D I F F E R E N T I A T I O N INDUCTION OF HL60 CELLS IN A LONG TERM BONE M A R R O W CULTURE OF ACUTE MYELOID LEUKEMIA G. J. OSSENKOPPELE,I. DENKERS, P. WIJERMANS, P. C. HUIJGENS, J. J. P. NAUTA,* R. J. H. BEELEN and M. M. A. C. LANGENHUIJSEN *Department of Haematology and Medical Statistics and Free University Hospital, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands
(Received 17 November 1989. Accepted 20 February 1990) Abstraet--Bone marrow stromal cells are critical for the proliferation and differentiation of hemopoietic stem cells. The hemopoietic microenvironment is reproduced in long term bone marrow culture (LTBMC). Normal LTBMC versus leukemic LTBMC and their stroma conditioned medium were compared with respect to their proliferative and differentiation-inducing capacities. Myeloid leukemic cells (HL60) were layered onto LTBMCs derived from normal volunteers and patients with AML. Differentiation was measured with a comprehensive panel of maturation parameters, i.e. morphology, cytochemistry, quantitative enzyme determination, NBT test and immunophenotyping. Inhibition of proliferation occurred in all cocultures. Clear maturation in monocytic direction was obvious in one culture of HL60 cells layered onto a leukemic stroma. As stroma-derived conditioned medium has no effect, a cellular interaction seems involved. These observations support not only the concept that normal stroma influences leukemic cell growth but also that leukemic stroma can modulate cell growth and maturation.
Key words: Differentiation, HL60 cells, long term bone marrow culture AML.
INTRODUCTION
kemia and from normal volunteers on leukemic cells with respect to proliferation and differentiation inducing capacities.
THE HEMOPOIETIC microenvironment plays a critical role in proliferation and differentiation of stem cells. Dexter et al. [1] developed a system for the long term culture of murine m a r r o w , which has been adapted to studies of h u m a n m a r r o w [2, 3, 4]. These long term bone m a r r o w cultures ( L T B M C ) are characterized by a stromal layer c o m p o s e d of fibroblasts, macrophages, fat cells and endothelial cells producing an extracellular matrix possibly responsible for microenvironment specificity [5, 6]. L T B M C has allowed the study of the h a e m o p o i e t i c microenvironment providing a model for the complex interactions between stroma cells, extracellular matrix and haemopoietic stem cells from normals as well as from patients with hematological disorders. We recently introduced a comprehensive panel of quantitative and qualitative maturation p a r a m e t e r s useful in the investigation of myeloid differentiation in cell lines and freshly obtained leukemic cells [7, 8]. In this study we examined the influence of stromal cells derived from patients with acute myeloid leu-
MATERIALS AND METHODS
Long term bone marrow culture (LTBMC) Bone marrow cells were obtained from two normal volunteers and three consecutive patients with acute myeloid leukemia (clinical data are summarized in Table 1). Mononuclear cells were isolated after centrifuging on Ficoll-Hypaque density gradient (d = 1.077) at 400g for 30 min and washed twice in Hank's-BSA 0.1%. Bone marrow mononuclear cells were suspended in LTBMC growth medium (alpha-MEM supplemented with 400 mg/liter glutamine, 40 mg/liter inositol, 10 mg/liter folic acid, 12.5% horse serum, 12.5% fetal calf serum, 10-6M hydrocortisone and antibiotics) to give a final concentration of 1 × 106/ml; 5 ml aliquots were added to 25 cm2 culture flasks (Falcon). The cultures were incubated at 37°C in humidified 5% CO2 and fed weekly by complete replacement of the LTBMC growth medium until the bottoms were covered by confluent layers of stromal cells and hemopoietic progenitors had disappeared. Stroma conditioned medium (SCM) Supernatants discarded before the LTBMCs were used for coculture experiments were collected and added to HL60 cells as described below.
Correspondence to: G. J. Ossenkoppele, Free University Hospital, Department of Haematology (BR238), De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. 611
612
G . J . OSSENKOPPELE et al.
Cell line The HL60 promyelocytic leukemia cell line was maintained in suspension culture in alpha-MEM (Gibco, Grand Island, New York) containing 20% fetal calf serum (FCS) (Flow Lab, Rockville, MD), heat inactivated at 56°C for 30 min, with penicillin and streptomycin. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in air. Coculture o f HL60 cells onto L T B M C and with SCM HL60 cells were seeded at a density of 1 × 106ce11/ ml onto LTBMC. After 96 h HL60 cells were harvested, attached cells were detached by gentle pipetting with PBS. Also HL60 cells were added to SCM at a density of 1 × 106 cell/ml and harvested after 4 days. HL60 control cells grew in LTBMC growth medium for 4 days. HL60 control cells grew in LTBMC growth medium for 4 days. Vitality of harvested cells was always >90% as determined by trypan blue dye exclusion. Cell counts Cell number was determined by flow cytometric technique on an AI Cell Counter 134 (Analysis Instruments). Cytochemistry After 96 h in culture cells were collected on slides using a Shandon cytocentrifuge. Cytospin slide preparations were stained according to May-Grunwald-Giemsa for assessment of morphology. The cells were also stained with Sudan black and alpha naphthyl acetate esterase stains as previously described [19]. Differential counting of at least 100 cells was performed by two members of our study group independently. In each cytochemical stain 100 cells were judged for positivity by at least two of the investigators. Intracellular enzyme activity Cells were lysed by freezing them three times in liquid nitrogen. Cell fragments were spun down at 7000g for 4 minutes, the collected supernatant was used for enzyme determination. Techniques included minor variations, as described in detail elsewhere [14], of standard methods for the estimation of myeloperoxidase [10], acid esterase using both alpha naphthyl acetate and alpha naphthyl butyrate as a substrate [11]. The amount of enzyme is expressed as units per 106 cells and as units per mg protein. The amount of protein in the cell lysate was determined according to the method of Lowry et al. [12]. Nitro blue tetrazolium ( N B T ) reduction The reduction of NBT was measured quantitatively. One hundred microliters of cells (0.5 x 106/ml) in tr-MEM containing 20% FCS were incubated at 37°C for 15 min with 100 ~tl NBT (0.5 mg/ml) and 2 ~tl 10 ~tM freshly diluted 12-O-tetradecanoyl phorbol 13-acetate (TPA). A native preparation was made in which the percent of cells containing blue black deposits of formazan was determined by evaluating 200 cells. Cell diameter Cell diameters were measured in cell suspensions by means of an Elzone 80XY Volume Counter (Particle, Data, U.S.A.), which measures and plots cell diameters according to the coulter principle in 128 channels.
TABLE 1. CLINICALDATA OF THREE PATIENTSWITH AML Patient Sex Age 1 2 3
f f m
81 30 52
FAB Cytogenetics M5b M4 M4
46 XX 46 XX (inv9) 46 XY
% Blasts
CR
92% 80% 89%
+ +
Immunophenotyping Cell membrane phenotypes were determined by using commercially available monoclonal antibodies combined with an immunoperoxidase technique. Antibodies included: HLA-Dr (Dakopatts, Denmark), E B M l l (Dakopatts, Denmark) [13] a monocyte-macropbage specific antibody and RFD7 (kindly provided by Dr L. W. Poulter) [14] a marker for mature macrophages. Statistical evaluation Data analysis was performed using Student's t-test. When the response variable was a proportion, the statistical analysis was not performed on the values themselves but on the values transformed by the angular transformation, A = arcsin(X/p). When proportions are close to either 0 (zero) or 100 the normality assumption applies better to these transformed values. The resulting confidence limits were transformed back to the original scale.
RESULTS The proliferation of H L 6 0 cells layered o n t o the normal L T B M C s as well as o n t o the leukemic L T B M C s was inhibited. A l s o g r o w t h in the presence of SCM of either origin was diminished. T h e s t r o m a conditioned media derived f r o m all L T B M C did not induce any significant change in the m e a s u r e d differentiation parameters. A f t e r coculture with normal L T B M C , no i m p o r t a n t m o r p h o l o g i c a l change was o b s e r v e d in H L 6 0 cells. N e i t h e r did quantitative e n z y m e d e t e r m i n a t i o n reveal a significant change in c o n t e n t of protein, myeloperoxidase or acid esterase. N o c h a n g e in antigenic determinants was measured. H o w e v e r , in the H L 6 0 cells cocultured with the leukemic s t r o m a layer of patient two, clear signs of m a t u r a t i o n were o b s e r v e d concurrent with inhibition of cell growth. As is shown in Figs l a and l b a decrease in nuclearcytoplasmic ratio, loss of nucleoli and d e v e l o p m e n t of vacuoli was exhibited pointing to m a t u r a t i o n in monocytic direction. T h e p e r c e n t a g e of Sudan black positive cells r e m a i n e d 100% t h o u g h intensity decreased. T h e strong positivity for non specific acid esterase was most striking (Figs 2a and 2b). These m o r p h o l o g i c findings are confirmed by a decrease in m y e l o p e r o x i d a s e content and an increment of acid esterase a m o u n t by using alpha naphthyl acetate as well as alpha n a p h t h y l butyrate as substrate. Antigenic d e t e r m i n a n t s were compatible
FIG. la. HL60 control cells after 4 days culture in medium (MGG-staining).
FIG. lb. HL60 cells after coculture onto long term bone marrow culture from patient two (MGG-staining). Monocytic differentiation is shown. 613
FIG. 2a. HL60 control cells after 4 days culture in medium (non specific esterase staining).
FIG. 2b. HL60 cells after coculture onto long term bone marrow from patient two. This non specific esterase staining is clearly positive. 614
Differentiation in a long term bone marrow culture
615
with a more mature celltype as shown by a moderate increase in H L A - D r positive cells and a remarkable increase in E M B l l positive cells. Coculturing HL60 cells with stroma of patients one and three did not lead to maturation.
< ©
DISCUSSION Z
