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An in vitro analysis of murine hemopoietic fibroblastoid progenitors and fibroblastoid cell function during aging
Mechanisms o f Ageing and Development, 22 (1983) 11 -21
11
Elsevier Scientific Publishers Ireland Ltd.
AN I N VITRO ANALYSIS OF MURINE HEMOPOIETIC FIBROBLASTOID PROGENITORS AND FIBROBLASTOID CELL FUNCTION DURING AGING
K.G.M. BROCKBANK*,R.E. PLOEMACHERand C.M.J. VAN PEER Department o f Cell Biology and Genetics, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam (The Ne therlands)
(Received October 6th, 1982) SUMMARY We determined the number of fibroblastoid progenitors (fibroblastoid colony-forming units, CFU-F) in femurs and spleens derived from (CBA × C57BL)F 1 mice of different ages. The femoral CFU-F population size increased from 350 at 1 week of age and plateaued at approximately 1900 CFU-F at 8 weeks of age. The mean incidence of CFU-F per 106 femoral marrow nucleated cells decreased from 82 at 8 weeks of age to 55 at 70 weeks of age; however, due to an increase in femur cellularity, there was no decrease in the CFU-F population size. The splenic CFU-F population decreased from 1700 at 1 week of age to 180 at 8 weeks of age; no further change was observed in mice up to 70 weeks' old. Analysis of colony-stimulating activity production by fibroblastoid colonies derived from young (6 weeks) and aged (70 weeks) mouse femoral marrow demonstrated no difference. These results indicate that there is no change in CFU-F numbers or fibroblastoid cell colony-stimulating activity production associated with the age-related increase in hemopoietic organ cellularity and hemopoietic progenitor content observed in this mouse strain. There were, however, major changes in the CFU-F population sizes during development of both femoral marrow and spleen in the first 2 months after birth.
Key
words: Aging; Fibroblasts; Microenvironment; Hemopoiesis; Colony-stimulating
activity
INTRODUC~ON The presence of fibroblastoid colony-forming units (CFU-F) in murine hemopoietic organs has been demonstrated by a number of investigators [ 1-4]. Friedenstein et al. [5 ] *To whom correspondence should be addressed. 0047-6374/83/$03.00 Printed and Published in Ireland
© 1983 ElsevierScientific Publishers Ireland Ltd.
12 showed that cultured guinea-pig fibroblastoid cells derived from bone marrow and spleen were capable of transferring the hemopoietic microenvironment characteristic of their organ origin. Bone marrow fibroblastoid cells have also been shown to produce a factor which increases survival of hemopoietic pluripotent stem cells (CFU-s) in vitro [6] and colony-stimulating activity (CSA) for granulocyte/macrophage progenitors [2,7]. During the first 7 weeks after birth an age-dependent decline in the incidence of CFU.F recoverable from routine bone marrow has been reported [3]. Similarly, an inverse correlation between donor age and the number of CFU-F in bone marrow has been demonstrated in humans [8]. In this report we have determined the femoral and splenic CFU-F population size in mice varying in aging. The results indicate that there are changes in CFU-F incidence associated with age, but that the absolute number of CFU-F per hemopoietic organ remains constant beyond the first 2 months after birth. In addition, the ability of fibroblastoid cells derived from the femoral marrow of young and aged mice to produce CSA is compared.
MATERIALS AND METHODS Animals Male (CBA × C57BL)F 1 mice were employed. The mice were killed by cervical dislocation prior to removal of both femurs and the spleen using sterile technique. All experiments, unless otherwise stated, were performed on cells from individual mice. Cell preparation Bone marrow was flushed from femurs with 6 ml of buffered salt solution (BSS) containing 5% fetal calf serum (FCS) after amputation of the epiphyses. Spleen cells were pressed through a nylon mesh sieve in 6 ml of BSS containing 5% FCS. Single-cell suspensions were then prepared by repeated flushing of the spleen or bone marrow cells through a number 23 gauge syringe needle. The cells were centrifuged and resuspended in BSS containing 5% FCS prior to dilution to the required cell concentration ha a-medium (a-modification of Dulbecco's minimum essential medium) containing 5% MCS. Nucleated cell counts were performed in a Coulter particle counter. Fibroblastoid cell culture Bone marrow and spleen cells were cultured in a-medium containing 0.8% methylcellulose and 20% FCS. Aliquots (1 ml) of ceils in culture medium were plated in 35-mm culture dishes and incubated at 37°C in an atmosphere consisting of 10% CO2 in air. All determinations were performed in triplicate. On day 10 of culture the dishes were washed with phosphate-buffered saline (PBS) fixed in methanol and stained with 10% Giemsa. Fibroblastoid colonies containing at least 50 fibroblastoid cells were counted with the aid of an inverted microscope.
13
Hemopoietic progenitor quantitation Granulocyte/macrophage colony-forming units (CFU-G/M) and erythroid burstforming units (BFU-E) were quantitated in a semisolid (0.8% methylcellulose) culture medium (a-medium) at 37°C in atmosphere consisting of 5% CO: in air. For both CFUG/M and BFU.E assays either between 5 × 104 and 10 × 104 bone marrt~w cells or 5 X l0 s spleen cells were plated. CFU-G/M cultures also contained 20% concanavalin A (Con-A)-stimulated mouse spleen conditioned medium (MSCM), 10% FCS and 1% bovine serum albumin (BSA). BFU-E cultures contained 20% Con-A stimulated MSCM, 10% FCS, 1% BSA, 10 -4 M mercaptoethanol, and 0.5 U of step III preparation of sheep plasma erythropoietin (Ep, Connaught Labs. Ltd., Willowdale, Ontario, Canada). Granulocyte/macrophage colonies and erythroid bursts were counted on day 7 and day 10 of culture, respectively, with an inverted microscope. All colonies and bursts counted contained at least 50 cells.
Spleen colony assay The CFU-s content of hemopoietic organs was determined by the spleen colony assay in lethally irradiated (850 rad) recipient mice. The recipient mice were killed 7 days later and their spleens fixed in Telleyesnizky's solution. The colonies were counted with the aid of a dissection microscope.
Double-layer cultures The ability of fibroblastoid cells to support the formation of granulocyte/macrophage colonies in the absence of an exogenous CSA was examined in double-layer cultures. Fibroblastoid colonies were established in 35-mm Costar tissue culture dishes and incubated at 37°C in an atmosphere consisting of 10% CO2 in air. The cultures were fed by the addition of 1.0 ml of a-medium with 20% FCS on day 7 of culture. On day 17 or 18 of culture the methylcellulose-containingmedium was removed. The subconfluent monolayers of fibroblastoid colonies were washed twice with PBS and then 1.0 ml of 0.3% bactoagar in a-medium with 20% FCS was placed on top of the fibroblastoid colonies. The establishment of the double-layer cultures was completed by placing 105 nucleated bone marrow cells, derived from 3 pooled lO-20-week-old mice, in 1.0 ml of a-medium containing 0.8% methylcellulose, 10% FCS and 1% BSA over the agar layer. The double-layer cultures were incubated at 37°C in an atmosphere consisting of 5% CO2 in air for 7 days, and colonies containing at least 50 granulocytes and/or macrophages were scored. RESULTS
Cellular composition of flbroblastoid colonies The major cell type present in adherent colonies derived from both bone marrow and spleen was a large polymorphic fibroblastoid cell with pale cytoplasm and an ovoid nucleus. Smaller macrophages were also present in some colonies and between colonies,
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Fig. 1. T i m e - c o u r s e study of femoral (a) and splenic (e) fibroblast colony formation in culture, Either 10 ~ bone marrow cells or 5 X 106 spleen cells, derived from 3 pooled ot-gans from 4 - 5 - w e e k old mice, were plated in triplicate. One group of dishes was removed for colony analysis each day on days 4 - 14. The data are presented as the mean -+ 1 S.E. of 4 experiments.
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Fig. 2. Linearity analysis of fibroblast colony formation with respect to the cell number plated. Bone marrow cells or spleen cells, derived from individual 4-5-week-old mice, were plated in triplicate. A linear relationship was observed for femoral CFU-F (a) between 2.5 × 105 and 10 × 105 nucleated cells per dish (correlation coefficient = 0.98, slope = 3.53) and for spleen CFU-F (e) between 2 X 10 a and 6 × 10 ~ nucleated cells per dish (correlation coefficient = 0.99, slope = 2.36). The data are presented as the mean +- 1 S.E. o f 6 experiments,
15 especially in cultures of spleen cells. The fibroblastoid cells and macrophages could be discriminated on the basis of their phagocytic activity and morphology [7,9].
Formation of fibroblastoid colonies In cultures established from bone marrow and spleen the number of fibroblastoid colonies containing at least 50 fibroblastoid cells increased until day 10 of culture and then plateaued (Fig. 1). Cultures were not followed further than day 14 because the colonies became confluent. On the basis of these results we counted fibroblastoid colonies on day 10 in all further experiments. A linear relationship was observed between the number of cells plated and the number of fibroblastoid colonies counted (Fig. 2). Bone marrow flbroblastoid colony formation was linear between 2.5 × l0 s and 10 × l0 s nucleated cells per dish (correlation coefficient = 0.98; slope = 3.53), whereas spleen fibroblastoid colony formation was linear between 2 × 10 6 and 6 X 10 6 nucleated cells per dish (correlation coefficient = 0.99, slope = 2.36). These observations permitted quantitative determinations of the incidence of CFU-F to be made in spleen and femoral marrow.
Age-related changes in the femoral CFU-Fpopulan'on The femoral CFU-F population doubled in size during the first 5 weeks of the study (Fig. 3); however, the total nucleated cell content per femur increased 5-fold (Table I) resulting in a 42% reduction of CFU-F per 106 nucleated cells (Fig. 3). The incidence of CFU-F then increased to 82 -+ 8 per 10 6 nucleated ceils (mean -+ 1 S.E.) at 8 weeks of age followed by a slow decrease to 55 + 7 per 106 nucleated cells at 70 weeks of age. This decrease in CFU-F incidence per 106 nucleated cells was associated with an increase in
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Fig. 3. Femoral CFU-F in mice varying in age. The data are presented as the mean ± 1 S.E. of 1 0 20 determinations on individual mice, either as CFU-F per 106 nucleated cells plated ( . ) or as CFU-F per femur (A).
16 TABLE I HEMOPOIETIC ORGAN CELLULARITY OF MICE VARYING IN AGE All data are expressed as the mean ± 1 S.E, of 1 0 - 2 0 individual mice.
Age (weeks)
Nucleated cells (X 10 ~) per organ
1 '~ 6 8 18 29 54 70
Femoral marrow
Spleen
0.48 1.94 2.35 2.41 2.63 2.54 3.68 3.70
4.49 17.98 19.50 17.96 20.36 18.85 22.21 30.15
± 0.06 ± 0.10 ± 0.09 ± 0.09 ± 0.11 ± 0.07 ± 0.10 ± 0.16
± 0.41 ± 0.96 ± 0.44 ± 0.78 ± 0.39 ± 0.48 ± 0.95 ± 2.18
f e m u r cellularity (Table I), w h i c h r e s u l t e d in n o overall c h a n g e in the f e m o r a l C F U - F p o p u l a t i o n size b e t w e e n the ages o f 8 a n d 70 weeks.
Age-related changes in the splenic CFU-F population The
incidence
o f C F U - F in spleen c u l t u r e s decreased rapidly f r o m
1 8 8 - - - 2 0 per
5 X 106 n u c l e a t e d cells ( m e a n +- 1 S.E.) at 1 w e e k o f age t o 15 -+ 2 p e r 5 × 106 n u c l e a t e d cells at 4 w e e k s o f age (Fig. 4). T h e C F U - F i n c i d e n c e stabilised at a p p r o x i m a t e l y 4 - 5
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Fig. 4. Splenic CFU-F in mice varying in age. The data are presented as the mean -+ 1 S.E. of 1 0 - 2 0 determinations on individual mice, either as CFU-F per 5 × 106 nucleated cells plated (o) or as CFU-F per spleen (').
4490±210 3570 ± 250 4 1 5 0 ± 90
15 6 0 0 ± 2 1 5 0 11 830 ± 1030 12 990 ± 1030
8010-+ 530 10960±2160
Spleen
Spleen
Femur
CFU-G/M per organ
CFU-s per organ
*Significantly different (p < 0.05) by Wilcoxon test.
8-12 36-44 85-104
Age (weeks)
Data axe presented as the mean ± 1 S.E. of 3 experiments on ceils pooled from 3 mice.
47 3 1 0 ± 3560 81 040 ± 2 1 6 0 "
Femur
9340 ± 1690 14 010 ± 2761
5 9 8 0 ± 1270
Femur 2185 ± 900
Spleen
BFU-E per organ
HEMOPOIETIC STEM CELL AND PROGENITOR CELL CONTENT OF HEMOPOIETIC ORGANS DERIVED FROM MICE V A R Y I N G IN AGE
TABLE II
18 per 5 × 106 nucleated spleen cells in mice of 18 weeks and older. Similarly, the absolute numbers of CFU.F in spleens decreased from 1700 +- 240 per spleen at 1 week of age to approximately 180 CFU-F per spleen in mice of 8 weeks and older. The period in which the splenic CFU-F population size decreased coincided with growth of the spleen (Table I).
Hemopoietic stem cells and progenitor cells The femoral CFU-G/M population size of aged mice was significantly higher (p < 0.05, Wilcoxon test) than in young mice and there was a general tendency for CFU-G/M and BFU-E to be higher in both spleen and femur of aged mice (Table II). No significant change in CFU-s content of hemopoietic organs was observed with age.
CSA production by fibroblastoid colonies The ability of young and aged femoral marrow fibroblastoid cells to support granulocyte/macrophage colony formation was compared. Femoral marrow cells were plated at cell concentrations below that at which we expected a linear relationship between nucleated cells plated and fibroblastoid colonies, i.e. less than 2 × lOs nucleated cells per dish. Dishes containing 0, 1, 2 and 3 fibroblastoid colonies were selected on day 17 or 18 of culture and double-layer cultures were established over them; granulocyte/ macrophage colonies which developed in the upper layer were observed above the fibroblastoid colonies 7 days later. A linear relationship between fibroblastoid colonies in the lower layer and granulocyte/macrophage colonies formed in the upper layer was obtained for fibroblastoid colonies derived from both young (6 week) and aged (70 week) femoral marrow, the correlation coefficients being 0.99 and 0.96, respectively. The data
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Fig. 5. The ability of young (6 week) marrow f~roblastoid colonies (o) and aged (70 week) marrow fibroblastoid colonies (.) to support the formation of gmnulocyte/macrophage colonies was compared in a double-layer culture system. The data are presented as the mean +- 1 S.E. of the number of dishes (enclosed in parentheses).
19 are presented in Fig. 5. There was no difference in the number of granulocyte/macrophage colonies developing in the presence of fibroblastoid colonies derived from young and aged murine marrow. There did not appear to be any qualitative size differences between fibroblastoid colonies derived from mice varying in age. DISCUSSION During the first 5 weeks of this study we observed an age-dependent decline in the incidence of CFU-F per 10 6 nucleated femoral marrow cells plated (Fig. 3). This observation is in agreement with the results presented by Zipori and van Bekkum [3] ;however, these authors did not estimate the total CFU-F population size of the femurs. When we analyzed the CFU-F content of femurs from mice aged 1-6 weeks it was apparent that there was an increase in femoral CFU-F population size during this period. The decline in CFU-F incidence per 10 6 nucleated cells plated was due to CFU-F dilution by hemopoietic cells during growth of the femur. The main expansion of the femoral CFU-F population occurred between 6 and 8 weeks of age. It is possible that the relatively slow increase in the femoral CFU-F population size in the first weeks of this study was due to a predominance of CFU-F differentiation to form microenvironments for hemopoiesis over CFU-F self-renewal. The femoral CFU-F population size plateaued at approximately 1900 CFU-F at week 8. Both the splenic CFU-F population size and incidence per 10 6 nucleated cells plated decreased rapidly during growth of the spleen and by the eighth week the splenic CFU-F population size had stabilised at approximately 180 CFU-F. No further change was observed in the CFU-F content of either hemopoietic organ with aging. In agreement with previous reports [ 10,11 ] we observed an increased absolute femoral cellularity in aged animals. Due to this increased femoral cellularity the incidence of CFU-F per 10 6 cells plated decreased from 82 + 8 at weeks of age to 55 + 7 at 70 weeks of age. Mets and Verdonk [8] observed a more dramatic decrease in CFU-F incidence and an increase in small (<32 cells) epithelial-like clones in human marrow aspirates. It is possible that the decrease in human marrow might not be so large if the data could be expressed per femur. We did not observe epithelial-like cells in cultures of murine hemopoietic stroma and there was no increased incidence of fibroblastoid clones containing fewer than 50 cells with aging. Our finding that there was a linear relationship between nucleated cells plated and the number of CFU-F detected is indirect evidence for the clonal origin of CFU-F (Fig. 2). Piersma e t al. [12] demonstrated the clonal origin of CFU-F in cultures of chimaeric bone marrow containing normal and T6 chromosome marked CFU-F; all countable mitoses in any one colony contained the same chromosome complement. We found no difference in CSA production by young (6 week) and aged (70 week) murine femoral marrow derived fibroblastoid colonies (Fig. 5). Fibroblastoid cells were present in the adherent layer of long-term (Dexter.type) bone marrow cultures in which long-term maintenance and self-renewal of hemopoietic stem cells have been demon-
20 strated [13,14]. Recently, Matthews and Crouse [15] found that Dexter-type cultures derived from aged marrow supported a greater CFU-s number and total nucleated cellularity than cultures established from young marrow. Similarly, an age-related increase in femoral CFU-s content has been observed in vivo in some mouse strains [11,16]. In the (CBA XC57BL)F1 mice employed in our experiments an age-related increase in hemopoietic organ cellularity (Table I) and hemopoietic organ progenitor content (Table II) was observed, but there was no increase in CFU-s numbers. This enhanced hemopoiesis in aged marrow did not correlate with either a numerical difference in CFU-F content or fibroblastoid cell function. The C'FU-F population size remained constant beyond the age of 8 weeks and fibroblastoid cell CSA production was the same in marrow derived from young and aged mice. ACKNOWLEDGEMENTS We would like to thank Prof. Dr. O. Vos for critically reviewing this manuscript and Mrs. Cary Meijerink-Clerkx for assistance in manuscript preparation. This investigation was supported by The Netherlands Foundation for Pure Research (ZWO).
REFERENCES 1 A.J. Friedenstein, U.F. Deriglasova, N.N. Kulagina, A.F. Panasuk, S.F. Rudakowa, E.A. Luria and I.A. Rudakow, Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp. Hematol., 2 (1974) 83-92. 2 F.D. Wilson, L. O'Grady, C.J. McNeiU and S.L. Munn, The formation of bone marrow derived fibroblastic plaques in vitro: preliminary results contrasting these populations to CFU-C. Exp. Hematol., 2 (1974) 343-354. 3 D. Zipori and D.W. van Bekkum, Changes in the fibrobiastoid colony-forming unit populations from mouse bone marrow in early stages of Soule virus induced murine leukemia. Exp. Hematol., 7 (1979) 137-143. 4 E.D. Werts, R.L. DeGowin, S.K. Knapp and D.P. Gibson, Characterization of marrow stromal (fibroblastoid) cells and their association with erythropoiesis. Exp. Hematol., 8 (1980) 423-433. 5 A.J. Friedenstein, R.K. Chailakhyan, N.B. Latsinik, A.F. Panasyuk and I.V. Keiliss-Borok,Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Transplantation, 17(1974) 331-340. 6 M.J. Blackburn and H.M. Patt, Increased survival of hemopoietic pluripotent stem cells in vitro induced by a marrow fibroblast factor. Br. J. Haematol., 37 (1977) 337-344. 7 K.G.M. Brockbank and C.M.J. van Peer, CSA production by hemopoietic organ fibroblastoid cells. Acta Haematol., 170 (1983) in press. 8 T. Mets and G. Verdonk, Variations in the stromal cell population of human bone marrow during aging. Mech. Ageing Dev., 15 (1981) 41-49. 9 D. Zipori and S. Bol, The role of f~roblastoid ceils and macrophages from mouse bone marrow in the in vitro growth promotion of haemopoietic tumour cells. Exp. Hematol., 7 (1979) 206-218. 10 R.E. Toya and M.L. Davis, Age-related changes in bone marrow hemopoiesis potential in mice. Biomedicine, 19 (1973) 244-247. 11 M.G. Chert, Age-related changes in hematopoietic stem cell populations of a long-lived hybrid mouse. J. Cell. PhysioL, 78 (1971) 225-232. 12 A.H. Piersma, R.E. Ploemacher and K.G.M. Brockbank, Transplantation of stromal progenitor cells (CFU-F) in mice. Exp. HematoL, 10 (Suppl. 11) (1982) 135 (abstract). 13 T.M. Dexter, T.D. Allen and L.G. Lajtha, Conditions controlling the proliferation of haemopoietic stem cells in vitro. J. Cell. Physiol., 91 (1977) 335-344.
21 14 S.A. Bentley and J.-M. Foidart, Some properties of marrow derived adherent cells in tissue culture. Blood, 56 (1980) 1006-1012. 15 K.I. Matthews and D.A. Crouse, An in vitro investigation of the hematopoietic microenvironment in young and aged mice.Mech. Ageing Dev., 17 (1981) 289-303. 16 V. Covelli and P. Metalli, A late effect of radiation on the haematopoietic stem cells of the mouse. Int. J. Radiat. Biol., 23 (1973) 83-89.
11
Elsevier Scientific Publishers Ireland Ltd.
AN I N VITRO ANALYSIS OF MURINE HEMOPOIETIC FIBROBLASTOID PROGENITORS AND FIBROBLASTOID CELL FUNCTION DURING AGING
K.G.M. BROCKBANK*,R.E. PLOEMACHERand C.M.J. VAN PEER Department o f Cell Biology and Genetics, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam (The Ne therlands)
(Received October 6th, 1982) SUMMARY We determined the number of fibroblastoid progenitors (fibroblastoid colony-forming units, CFU-F) in femurs and spleens derived from (CBA × C57BL)F 1 mice of different ages. The femoral CFU-F population size increased from 350 at 1 week of age and plateaued at approximately 1900 CFU-F at 8 weeks of age. The mean incidence of CFU-F per 106 femoral marrow nucleated cells decreased from 82 at 8 weeks of age to 55 at 70 weeks of age; however, due to an increase in femur cellularity, there was no decrease in the CFU-F population size. The splenic CFU-F population decreased from 1700 at 1 week of age to 180 at 8 weeks of age; no further change was observed in mice up to 70 weeks' old. Analysis of colony-stimulating activity production by fibroblastoid colonies derived from young (6 weeks) and aged (70 weeks) mouse femoral marrow demonstrated no difference. These results indicate that there is no change in CFU-F numbers or fibroblastoid cell colony-stimulating activity production associated with the age-related increase in hemopoietic organ cellularity and hemopoietic progenitor content observed in this mouse strain. There were, however, major changes in the CFU-F population sizes during development of both femoral marrow and spleen in the first 2 months after birth.
Key
words: Aging; Fibroblasts; Microenvironment; Hemopoiesis; Colony-stimulating
activity
INTRODUC~ON The presence of fibroblastoid colony-forming units (CFU-F) in murine hemopoietic organs has been demonstrated by a number of investigators [ 1-4]. Friedenstein et al. [5 ] *To whom correspondence should be addressed. 0047-6374/83/$03.00 Printed and Published in Ireland
© 1983 ElsevierScientific Publishers Ireland Ltd.
12 showed that cultured guinea-pig fibroblastoid cells derived from bone marrow and spleen were capable of transferring the hemopoietic microenvironment characteristic of their organ origin. Bone marrow fibroblastoid cells have also been shown to produce a factor which increases survival of hemopoietic pluripotent stem cells (CFU-s) in vitro [6] and colony-stimulating activity (CSA) for granulocyte/macrophage progenitors [2,7]. During the first 7 weeks after birth an age-dependent decline in the incidence of CFU.F recoverable from routine bone marrow has been reported [3]. Similarly, an inverse correlation between donor age and the number of CFU-F in bone marrow has been demonstrated in humans [8]. In this report we have determined the femoral and splenic CFU-F population size in mice varying in aging. The results indicate that there are changes in CFU-F incidence associated with age, but that the absolute number of CFU-F per hemopoietic organ remains constant beyond the first 2 months after birth. In addition, the ability of fibroblastoid cells derived from the femoral marrow of young and aged mice to produce CSA is compared.
MATERIALS AND METHODS Animals Male (CBA × C57BL)F 1 mice were employed. The mice were killed by cervical dislocation prior to removal of both femurs and the spleen using sterile technique. All experiments, unless otherwise stated, were performed on cells from individual mice. Cell preparation Bone marrow was flushed from femurs with 6 ml of buffered salt solution (BSS) containing 5% fetal calf serum (FCS) after amputation of the epiphyses. Spleen cells were pressed through a nylon mesh sieve in 6 ml of BSS containing 5% FCS. Single-cell suspensions were then prepared by repeated flushing of the spleen or bone marrow cells through a number 23 gauge syringe needle. The cells were centrifuged and resuspended in BSS containing 5% FCS prior to dilution to the required cell concentration ha a-medium (a-modification of Dulbecco's minimum essential medium) containing 5% MCS. Nucleated cell counts were performed in a Coulter particle counter. Fibroblastoid cell culture Bone marrow and spleen cells were cultured in a-medium containing 0.8% methylcellulose and 20% FCS. Aliquots (1 ml) of ceils in culture medium were plated in 35-mm culture dishes and incubated at 37°C in an atmosphere consisting of 10% CO2 in air. All determinations were performed in triplicate. On day 10 of culture the dishes were washed with phosphate-buffered saline (PBS) fixed in methanol and stained with 10% Giemsa. Fibroblastoid colonies containing at least 50 fibroblastoid cells were counted with the aid of an inverted microscope.
13
Hemopoietic progenitor quantitation Granulocyte/macrophage colony-forming units (CFU-G/M) and erythroid burstforming units (BFU-E) were quantitated in a semisolid (0.8% methylcellulose) culture medium (a-medium) at 37°C in atmosphere consisting of 5% CO: in air. For both CFUG/M and BFU.E assays either between 5 × 104 and 10 × 104 bone marrt~w cells or 5 X l0 s spleen cells were plated. CFU-G/M cultures also contained 20% concanavalin A (Con-A)-stimulated mouse spleen conditioned medium (MSCM), 10% FCS and 1% bovine serum albumin (BSA). BFU-E cultures contained 20% Con-A stimulated MSCM, 10% FCS, 1% BSA, 10 -4 M mercaptoethanol, and 0.5 U of step III preparation of sheep plasma erythropoietin (Ep, Connaught Labs. Ltd., Willowdale, Ontario, Canada). Granulocyte/macrophage colonies and erythroid bursts were counted on day 7 and day 10 of culture, respectively, with an inverted microscope. All colonies and bursts counted contained at least 50 cells.
Spleen colony assay The CFU-s content of hemopoietic organs was determined by the spleen colony assay in lethally irradiated (850 rad) recipient mice. The recipient mice were killed 7 days later and their spleens fixed in Telleyesnizky's solution. The colonies were counted with the aid of a dissection microscope.
Double-layer cultures The ability of fibroblastoid cells to support the formation of granulocyte/macrophage colonies in the absence of an exogenous CSA was examined in double-layer cultures. Fibroblastoid colonies were established in 35-mm Costar tissue culture dishes and incubated at 37°C in an atmosphere consisting of 10% CO2 in air. The cultures were fed by the addition of 1.0 ml of a-medium with 20% FCS on day 7 of culture. On day 17 or 18 of culture the methylcellulose-containingmedium was removed. The subconfluent monolayers of fibroblastoid colonies were washed twice with PBS and then 1.0 ml of 0.3% bactoagar in a-medium with 20% FCS was placed on top of the fibroblastoid colonies. The establishment of the double-layer cultures was completed by placing 105 nucleated bone marrow cells, derived from 3 pooled lO-20-week-old mice, in 1.0 ml of a-medium containing 0.8% methylcellulose, 10% FCS and 1% BSA over the agar layer. The double-layer cultures were incubated at 37°C in an atmosphere consisting of 5% CO2 in air for 7 days, and colonies containing at least 50 granulocytes and/or macrophages were scored. RESULTS
Cellular composition of flbroblastoid colonies The major cell type present in adherent colonies derived from both bone marrow and spleen was a large polymorphic fibroblastoid cell with pale cytoplasm and an ovoid nucleus. Smaller macrophages were also present in some colonies and between colonies,
14
35
30
5 25
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0 ne u.
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Fig. 1. T i m e - c o u r s e study of femoral (a) and splenic (e) fibroblast colony formation in culture, Either 10 ~ bone marrow cells or 5 X 106 spleen cells, derived from 3 pooled ot-gans from 4 - 5 - w e e k old mice, were plated in triplicate. One group of dishes was removed for colony analysis each day on days 4 - 14. The data are presented as the mean -+ 1 S.E. of 4 experiments.
40 "r u')
c-, 3o z o 0 u
20
o
,n
10
2
4 6 NUCLEATED CELLS PLATED
8
10
Fig. 2. Linearity analysis of fibroblast colony formation with respect to the cell number plated. Bone marrow cells or spleen cells, derived from individual 4-5-week-old mice, were plated in triplicate. A linear relationship was observed for femoral CFU-F (a) between 2.5 × 105 and 10 × 105 nucleated cells per dish (correlation coefficient = 0.98, slope = 3.53) and for spleen CFU-F (e) between 2 X 10 a and 6 × 10 ~ nucleated cells per dish (correlation coefficient = 0.99, slope = 2.36). The data are presented as the mean +- 1 S.E. o f 6 experiments,
15 especially in cultures of spleen cells. The fibroblastoid cells and macrophages could be discriminated on the basis of their phagocytic activity and morphology [7,9].
Formation of fibroblastoid colonies In cultures established from bone marrow and spleen the number of fibroblastoid colonies containing at least 50 fibroblastoid cells increased until day 10 of culture and then plateaued (Fig. 1). Cultures were not followed further than day 14 because the colonies became confluent. On the basis of these results we counted fibroblastoid colonies on day 10 in all further experiments. A linear relationship was observed between the number of cells plated and the number of fibroblastoid colonies counted (Fig. 2). Bone marrow flbroblastoid colony formation was linear between 2.5 × l0 s and 10 × l0 s nucleated cells per dish (correlation coefficient = 0.98; slope = 3.53), whereas spleen fibroblastoid colony formation was linear between 2 × 10 6 and 6 X 10 6 nucleated cells per dish (correlation coefficient = 0.99, slope = 2.36). These observations permitted quantitative determinations of the incidence of CFU-F to be made in spleen and femoral marrow.
Age-related changes in the femoral CFU-Fpopulan'on The femoral CFU-F population doubled in size during the first 5 weeks of the study (Fig. 3); however, the total nucleated cell content per femur increased 5-fold (Table I) resulting in a 42% reduction of CFU-F per 106 nucleated cells (Fig. 3). The incidence of CFU-F then increased to 82 -+ 8 per 10 6 nucleated ceils (mean -+ 1 S.E.) at 8 weeks of age followed by a slow decrease to 55 + 7 per 106 nucleated cells at 70 weeks of age. This decrease in CFU-F incidence per 106 nucleated cells was associated with an increase in
i
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2000
150
1500 i
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500
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2~0
3~0 ;0 AGE OF MICE (WEEKS)
s~O
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70
Fig. 3. Femoral CFU-F in mice varying in age. The data are presented as the mean ± 1 S.E. of 1 0 20 determinations on individual mice, either as CFU-F per 106 nucleated cells plated ( . ) or as CFU-F per femur (A).
16 TABLE I HEMOPOIETIC ORGAN CELLULARITY OF MICE VARYING IN AGE All data are expressed as the mean ± 1 S.E, of 1 0 - 2 0 individual mice.
Age (weeks)
Nucleated cells (X 10 ~) per organ
1 '~ 6 8 18 29 54 70
Femoral marrow
Spleen
0.48 1.94 2.35 2.41 2.63 2.54 3.68 3.70
4.49 17.98 19.50 17.96 20.36 18.85 22.21 30.15
± 0.06 ± 0.10 ± 0.09 ± 0.09 ± 0.11 ± 0.07 ± 0.10 ± 0.16
± 0.41 ± 0.96 ± 0.44 ± 0.78 ± 0.39 ± 0.48 ± 0.95 ± 2.18
f e m u r cellularity (Table I), w h i c h r e s u l t e d in n o overall c h a n g e in the f e m o r a l C F U - F p o p u l a t i o n size b e t w e e n the ages o f 8 a n d 70 weeks.
Age-related changes in the splenic CFU-F population The
incidence
o f C F U - F in spleen c u l t u r e s decreased rapidly f r o m
1 8 8 - - - 2 0 per
5 X 106 n u c l e a t e d cells ( m e a n +- 1 S.E.) at 1 w e e k o f age t o 15 -+ 2 p e r 5 × 106 n u c l e a t e d cells at 4 w e e k s o f age (Fig. 4). T h e C F U - F i n c i d e n c e stabilised at a p p r o x i m a t e l y 4 - 5
i
200
2000
150
1500
100
1000
50
500
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-
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!
10
20
e 30 /..~0 AGE OF" MICE (WEEKS)
'11
i
0
50
6~0
70
Fig. 4. Splenic CFU-F in mice varying in age. The data are presented as the mean -+ 1 S.E. of 1 0 - 2 0 determinations on individual mice, either as CFU-F per 5 × 106 nucleated cells plated (o) or as CFU-F per spleen (').
4490±210 3570 ± 250 4 1 5 0 ± 90
15 6 0 0 ± 2 1 5 0 11 830 ± 1030 12 990 ± 1030
8010-+ 530 10960±2160
Spleen
Spleen
Femur
CFU-G/M per organ
CFU-s per organ
*Significantly different (p < 0.05) by Wilcoxon test.
8-12 36-44 85-104
Age (weeks)
Data axe presented as the mean ± 1 S.E. of 3 experiments on ceils pooled from 3 mice.
47 3 1 0 ± 3560 81 040 ± 2 1 6 0 "
Femur
9340 ± 1690 14 010 ± 2761
5 9 8 0 ± 1270
Femur 2185 ± 900
Spleen
BFU-E per organ
HEMOPOIETIC STEM CELL AND PROGENITOR CELL CONTENT OF HEMOPOIETIC ORGANS DERIVED FROM MICE V A R Y I N G IN AGE
TABLE II
18 per 5 × 106 nucleated spleen cells in mice of 18 weeks and older. Similarly, the absolute numbers of CFU.F in spleens decreased from 1700 +- 240 per spleen at 1 week of age to approximately 180 CFU-F per spleen in mice of 8 weeks and older. The period in which the splenic CFU-F population size decreased coincided with growth of the spleen (Table I).
Hemopoietic stem cells and progenitor cells The femoral CFU-G/M population size of aged mice was significantly higher (p < 0.05, Wilcoxon test) than in young mice and there was a general tendency for CFU-G/M and BFU-E to be higher in both spleen and femur of aged mice (Table II). No significant change in CFU-s content of hemopoietic organs was observed with age.
CSA production by fibroblastoid colonies The ability of young and aged femoral marrow fibroblastoid cells to support granulocyte/macrophage colony formation was compared. Femoral marrow cells were plated at cell concentrations below that at which we expected a linear relationship between nucleated cells plated and fibroblastoid colonies, i.e. less than 2 × lOs nucleated cells per dish. Dishes containing 0, 1, 2 and 3 fibroblastoid colonies were selected on day 17 or 18 of culture and double-layer cultures were established over them; granulocyte/ macrophage colonies which developed in the upper layer were observed above the fibroblastoid colonies 7 days later. A linear relationship between fibroblastoid colonies in the lower layer and granulocyte/macrophage colonies formed in the upper layer was obtained for fibroblastoid colonies derived from both young (6 week) and aged (70 week) femoral marrow, the correlation coefficients being 0.99 and 0.96, respectively. The data
15"[3
8 lo-
g o o E ~u
5-
_0 o
e(231 i 0
i 1
i 2
Fibroblost colonies per d i s h
Fig. 5. The ability of young (6 week) marrow f~roblastoid colonies (o) and aged (70 week) marrow fibroblastoid colonies (.) to support the formation of gmnulocyte/macrophage colonies was compared in a double-layer culture system. The data are presented as the mean +- 1 S.E. of the number of dishes (enclosed in parentheses).
19 are presented in Fig. 5. There was no difference in the number of granulocyte/macrophage colonies developing in the presence of fibroblastoid colonies derived from young and aged murine marrow. There did not appear to be any qualitative size differences between fibroblastoid colonies derived from mice varying in age. DISCUSSION During the first 5 weeks of this study we observed an age-dependent decline in the incidence of CFU-F per 10 6 nucleated femoral marrow cells plated (Fig. 3). This observation is in agreement with the results presented by Zipori and van Bekkum [3] ;however, these authors did not estimate the total CFU-F population size of the femurs. When we analyzed the CFU-F content of femurs from mice aged 1-6 weeks it was apparent that there was an increase in femoral CFU-F population size during this period. The decline in CFU-F incidence per 10 6 nucleated cells plated was due to CFU-F dilution by hemopoietic cells during growth of the femur. The main expansion of the femoral CFU-F population occurred between 6 and 8 weeks of age. It is possible that the relatively slow increase in the femoral CFU-F population size in the first weeks of this study was due to a predominance of CFU-F differentiation to form microenvironments for hemopoiesis over CFU-F self-renewal. The femoral CFU-F population size plateaued at approximately 1900 CFU-F at week 8. Both the splenic CFU-F population size and incidence per 10 6 nucleated cells plated decreased rapidly during growth of the spleen and by the eighth week the splenic CFU-F population size had stabilised at approximately 180 CFU-F. No further change was observed in the CFU-F content of either hemopoietic organ with aging. In agreement with previous reports [ 10,11 ] we observed an increased absolute femoral cellularity in aged animals. Due to this increased femoral cellularity the incidence of CFU-F per 10 6 cells plated decreased from 82 + 8 at weeks of age to 55 + 7 at 70 weeks of age. Mets and Verdonk [8] observed a more dramatic decrease in CFU-F incidence and an increase in small (<32 cells) epithelial-like clones in human marrow aspirates. It is possible that the decrease in human marrow might not be so large if the data could be expressed per femur. We did not observe epithelial-like cells in cultures of murine hemopoietic stroma and there was no increased incidence of fibroblastoid clones containing fewer than 50 cells with aging. Our finding that there was a linear relationship between nucleated cells plated and the number of CFU-F detected is indirect evidence for the clonal origin of CFU-F (Fig. 2). Piersma e t al. [12] demonstrated the clonal origin of CFU-F in cultures of chimaeric bone marrow containing normal and T6 chromosome marked CFU-F; all countable mitoses in any one colony contained the same chromosome complement. We found no difference in CSA production by young (6 week) and aged (70 week) murine femoral marrow derived fibroblastoid colonies (Fig. 5). Fibroblastoid cells were present in the adherent layer of long-term (Dexter.type) bone marrow cultures in which long-term maintenance and self-renewal of hemopoietic stem cells have been demon-
20 strated [13,14]. Recently, Matthews and Crouse [15] found that Dexter-type cultures derived from aged marrow supported a greater CFU-s number and total nucleated cellularity than cultures established from young marrow. Similarly, an age-related increase in femoral CFU-s content has been observed in vivo in some mouse strains [11,16]. In the (CBA XC57BL)F1 mice employed in our experiments an age-related increase in hemopoietic organ cellularity (Table I) and hemopoietic organ progenitor content (Table II) was observed, but there was no increase in CFU-s numbers. This enhanced hemopoiesis in aged marrow did not correlate with either a numerical difference in CFU-F content or fibroblastoid cell function. The C'FU-F population size remained constant beyond the age of 8 weeks and fibroblastoid cell CSA production was the same in marrow derived from young and aged mice. ACKNOWLEDGEMENTS We would like to thank Prof. Dr. O. Vos for critically reviewing this manuscript and Mrs. Cary Meijerink-Clerkx for assistance in manuscript preparation. This investigation was supported by The Netherlands Foundation for Pure Research (ZWO).
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21 14 S.A. Bentley and J.-M. Foidart, Some properties of marrow derived adherent cells in tissue culture. Blood, 56 (1980) 1006-1012. 15 K.I. Matthews and D.A. Crouse, An in vitro investigation of the hematopoietic microenvironment in young and aged mice.Mech. Ageing Dev., 17 (1981) 289-303. 16 V. Covelli and P. Metalli, A late effect of radiation on the haematopoietic stem cells of the mouse. Int. J. Radiat. Biol., 23 (1973) 83-89.