Mechanisms of Ageing and Development, 54 (1990) 131--142
131
ElsevierScientificPublishers Ireland Ltd.
STROMAL H E M O P O I E T I C M I C R O E N V I R O N M E N T IN A G I N G
A.V. SIDORENKO, BUTENKO
L.F. ANDRIANOVA*,
T.V. MACSYUK AND G.M.
Laboratory of Pathophysiology, Institute of Gerontology of the Academy of Medical Sciences of the U.S.S.R., Vyshgorodskaya St. 67, 252655, Kiev 114 (U.S.S.R.)
(ReceivedAugust 29th, 1989) (RevisionreceivedDecember I st, 1989)
SUMMARY The bone marrow content, proliferative potential and proliferative activity of precursor cells for stromal fibroblast colony forming cells (CFC-F) were investigated in young and old CBA mice. The relationship between CFC-F and the number of bone marrow nucleated cells and granulocytic-macrophagal precursors (GM-CFC) was studied as well. The results obtained showed increased CFC-F contents in old animals. The proliferative potential of old mice CFC-F did not appear to differ from that of young animals. The proliferative activity of bone marrow CFC-F and hemopoietic stem cells - - spleen colony forming cells (CFC-S) was studied by determining the sensitivity to hydroxyurea administration. The responses were almost the same in young and old mice. A direct correlation between CFC-F and nucleated cells and GM-CFC precursors was found in young mice, but not in the old animals. The results of the present study have pointed to the reorganisation o f the stromal tissue microenvironment in bone marrow in old age.
K e y words: Bone marrow; Hemopoietic and stromal precursor cells; Aging
INTRODUCTION Age-related variations o f the lymphohemopoietic system, especially of its lymphoid part, are well documented [1--3]. Nevertheless, the mechanisms of these changes remain unclear. In the most general sense the problem is to distinguish the extrinsic (macro-, microenvironment) and intrinsic (mainly, genetic program) factors, modulating the hemopoietic and lymphoid cell functions during aging. *To whom all correspondenceshould be addressed. 0047-6374/90/$03.50 Printed and Published in Ireland
© 1990ElsevierScientificPublishers Ireland Ltd.
132 The stromal tissue seems to be one of the most important sources of extrinsic signals, which can cause the age-dependent changes in lymphohemopoiesis [4--6]. In this connection it was interesting to assess the functional organisation and activity of the lymphohemopoietic stromal microenvironment in aging. For this purpose, the experimental approach that involved the quantitation of stromal precursor cells (CFC-F) was used. The above cells provided clonal growth of fibroblast colonies in primary monolayer cultures o f lymphoid and hemopoietic tissues [7]. CFC-F occupy the central place in a stromal cell hierarchy responsible for transfer of the hemopoietic microenvironment and serve as "stromal stem cells" [7]. The experimental attempts to evaluate the CFC-F contents in bone marrow of young and old laboratory rodents and humans have yielded contradictory results [8--12]. In the present study we have compared the contents and some proliferative characteristics of the femoral CFC-F in young and old CBA mice. MATERIALSAND METHODS
Animals Female CBA mice (Stolbovaya Breeding Laboratory, U.S.S.R. Academy o f Medical Sciences) were used when 2--3 months (young) and 22--26 months (old) of age. Only healthy appearing mice found to be grossly free o f disease at autopsy were evaluated. Outbred guinea pigs of both sexes were used as a source o f bone marrow feeder cells in CFC-F cloning experiments.
Preparation of cell suspensions Femurs were removed aseptically, epiphyses were removed and bone marrow was flushed into ice-cold RPMI-1640 media (Flow Laboratories, Glasgow, Scotland). To prepare single cell suspension cells were passed repeatedly through syringe with needles o f various diameters. The obtained suspensions were employed for count of nucleated cells and for cloning hemopoietic and stromal precursor cells.
Hemopoietic cells (1) Femoral bone marrow nucleated cells were calculated in a hemocytometer using 3 °7o acetic acid solution. (2) Hemopoietic stem cells (CFC-S) were determined according to their ability to form colonies in spleens of lethally irradiated mice [13]. Young (3 months) irradiated (10 Gy) recipient mice were injected with 0.75 x 105 bone marrow cells from young or old donors. The recipients were killed at day 8 after grafting and the number of macroscopic hemopoietic colonies corresponding to the number of CFC-S was counted on the spleen surface. (3) The granulocyte-macrophage colony-forming cells (GM-CFC) were estimated
133 using the agar cultures of bone marrow cells [14]. Medium conditioned by spleen cells incubated for 72 h in the presence o f 5 mkg/ml Concanavalin A (Calbiochem, San Diego, CA) was used as a source o f colony-stimulating factor (CSF) [15]. 105 bone marrow cells were cultivated in supplemented McCoy's 5A medium (Serva, Heidelberg, F.R.G.) until day 8 when the microscopic colonies growing in cultures were scored [16].
Fibroblast colony-forming cells Femoral bone marrow was assayed for the content o f fibroblast colony-forming cells using the method described in detail elsewhere [16]. Briefly, murine bone marrow cells at starting plating density of 2.5 X 105 cells/cm 2 were cultivated along with irradiated (45 Gy) guinea pig bone marrow cells (3.5 X 105 cells/cm z) in 85070 RPMI 1640 medium with 15 070 o f fetal calf serum (Flow Laboratories, Glasgow, Scotland). The cultures were incubated for 12 days at 37°C in a humidified atmosphere of 10070 CO 2 and 9007o o f room air. At the end o f incubation, the cultures were fixed with 96°70 ethanol, stained with azur-eosin and examined under dissection microscope to score colonies consisting of at least 50 fibroblast cells.
Proliferative activity of CFC-F and CFC-S Hydroxyurea (Serva, Heidelberg, F.R.G.) that can eliminate selectively cells in Sphase of the cellular cycle was injected intraperitoneally in the dose of 1 mg/kg of body weight twice with a 7-h interval to the young and old mice. Sixteen to eighteen hours following the last injection the numbers of nucleated cells, CFC-F and CFC-S were determined in the femoral bone marrow o f the animals.
Statistics Student's t-test was used to evaluate all the data. RESULTS As seen in Table I, the efficiency of stromal fibroblast colony formation by bone marrow cells in the old mice was almost two times higher compared to the young animals. The absolute number of CFC-F in bone marrow from old animals was also increased due to significantly increased nucleated cell numbers in the bone marrow during aging (Table I). There were similar observations during explantation into culture of cells from individual donors or when bone marrow ceils from several donors were pooled. It should be stressed that there were no significant differences in average CFC-F values in age-matched animals when the above two approaches were applied. The linear relationship was found between the number of bone marrow cells being explanted into culture and the number o f grown fibroblast colonies (Fig. 1). Such a relationship was characteristic o f both young and old cells.
134
TABLE I THE NUMBER OF N U C L E A T E D CELLS A N D CFC-F IN BONE MARROW FROM YOUNG AND OLD MICE
Age
Method o f testing
No. o f experiments
No. o f nucleated cells in the f e m u r ( X IO5)
No. o f CFC-F in explanted cells, 10~
No. o f CFC-F in bone marrow o f the femur
Young
Inand individual mouse InapooP Inan individual mouse Inapool
11
131.45 ±
2.24 -+ 0.23
302.27 + 36.76
16 16
141.13 ± 10.64 200.38 ± 11.84"**
2.48 ± 0.31 4.40 - 0.46**
326.00:1:36.01 808.94 ± 87.10"**
15
201.73 ± 17.38"*
3.63 ± 0.30*
704.13 :l: 61.04"**
Old
7.37
The differences of corresponding indices between young and old mice are statistically significant: *P < 0.05; * * P < 0.01; and * * * P < 0.001. aTwo or three mice were used in each experiment if bone marrow cells were pooled.
For comparison of the proliferative potential of CFC-F in bone marrow of the young and old mice, the experiments were set up as paired (young/old) observations. The average cellularity of colonies was found to be almost the same in the young and old animals (Fig. 2).
200-
0~~
o u
100.
Jo
0
u. o
z
0
NO. OF
CELLS
P L A T E D ('106)
Fig. I. The linear relationship between the number of fibroblast colonies and the number of explanted bone marrow cells for young (O - - - O; r = 0.906; y = 2.92x + 42.78; P < 0.01 and old ( e - - e ; r = 0.948; y = 3.19x + 39.13) mice. The data are presented as the means of triplicate cultures from three different experiments.
135
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O >Z O
g,
2-
U
u.I D.
| o•
1 0
g
m
u.. 0
6 Z
young
old
Fig. 2. Fibroblast colony cellularity in bone marrow cultures from young and old mice. At the end of incubation (at day 12), the cultures were fixed and stained and the number of fibroblasts in the grown colonies was counted, using a dissection microscope. At least 52 colonies from young and 50 colonies from old animals were used. Mean cellularity values were 532 ± 46 and 566 ± 58, respectively.
TABLE II THE EFFECT OF IN VITRO A D M I N I S T E R E D H Y D R O X Y U R E A ON THE NUMBER OF NUCLEATED CELLS, CFC-F A N D CFC-S IN BONE MARROW OF YOUNG AND OLD MICE
Age
Hydroxyurea administration
No. o f nucleated cells in the f e m u r ( x 10~)
No. o f CFC-F: In explanted cells, 10~
In bone marrow In bone o f the f e m u r marrow 10~cells
In bone marrow o f the femur
Young
--
119 _ 13
2.30 __ 0.17
309 ± 29
17.0 __ 1.4
1615 _+ 356
Old
+ -+
47 ± 205 ± 94 ±
3.90 ± 0.36*** 3.90 __ 0.16 4.70 ± 0.20**
152 -+ 13"** 775 ± 25 389 ± 24***
16.3 _+ 1.6 12.7 "4" l.O 13.0 _+ 4.7
735 ± 128" 2564 - 167 1146 ± 429**
9*** 6*** 6***
No. o f CFC-S:
The results are mean values of bone marrow cellularity and CFC-S contents for 5 experiments and of CFC-F cloning for three experiments. The differences of indices between control and experimental (hydroxyurea treatment) animals of the same age are signficant: *P < 0.05; **P < 0.01; and ***P < 0.001.
136
To evaluate the proliferative activity, the parallel study was undertaken on the effect of in vitro administration of hydroxyurea on stromal (CFC-F) and hemopoietic (CFC-S) precursor cells from bone marrow of young and old mice. The data of these experiments are presented in Table II. After hydroxyurea injection, the fibroblast colony forming efficiency o f bone marrow cells increased in cultures from young and old animals alike (Fig. 3). At the same time, the hemopoietic colony forming efficiency did not change significantly in either animal group (See Table II). Because o f the hydroxyurea-induced decrease in the total nucleated cell numbers of the bone marrow, its contents o f the precursor cells, both stromal and hemopoietic, decreased considerably. It is noteworthy that bone marrow cellularity and its CFC-F and CFC-S contents in young and old mice reduced in about the same manner. For example, the number of nucleated cells in femoral bone marrow decreased to 40 _+ 8070 in young and 46 _.+ 307o in old animals (P > 0.05). The number of CFC-S decreased to 38 ___ 9070 and 45 _.+ 6070 (P > 0.05), respectively, while the number of CFC-F decreased to 49 _+ 4070and 50 _.+ 3070 ( P > 0.05), respectively. We then tried to find out if there existed a correlation between the bone marrow contents of CFC-F and hemopoietic cells - - nucleated cells and GM-CFC. Again, we found a substantial increase with age in the number of nucleated cells and CFC-F (Table III). As regards GM-CFC, we failed to detect any meaningful character of increase in the precursor cells in old animals, probably, because of the varying
$
4
Fig. 3. The number of fibroblasts from bone marrows of young (1,2) and old (3,4) mice, control (1,3) and in vivo administered hydroxyurea (2,4).
137 T A B L E III T H E RESULTS O F T H E P A R A L L E L ASSESSMENT OF T H E N U M B E R OF H E M O P O I E T I C A N D S T R O M A L P R E C U R S O R C E L L S IN T H E F E M O R A L BONE M A R R O W F R O M Y O U N G A N D O L D MICE
Indices
Young mice (n = 12)
No. o f the nucleated cells (105) No. o f G M - C F C per 105 o f the explanted cells No. of G M - C F C in whole bone marrow o f the femur No. o f C F C - F per 10~ o f the explanted cells No. o f CFC-F in whole bone marrow of the femur
mice 14)
Old
(n
=
189 3: 13'** 103 3 : 1 9
124 3 : 1 0 99 3 : 1 6 14 399 3:2849
20 333 3:4156
2.48 3:0.28
4.55 3: 0.58***
3 I0 3 : 4 3
811 _+ 103
The differences of corresponding indices between y o u n g and old mice are statistically significant: ***P < 0.001.
cloning efficiency from one experiment to another. The number of CFC-F appeared to correlate positively both with the total number of hemopoietic (nucleated) cells, and with the number of GM-CFC in the young mice but not in the old animals (Fig. 4). o
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,~
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CFC-F
0
I
2 per
femur
(.lO
I
4
I
6
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8
110
112
I
14
2)
Fig. 4. The relationships between C F C - F and the total n u m b e r of nucleated cells (a) or granulocyte/ macrophage colonies per femur (b) in y o u n g (O - - O) and old ( e - - e ) mice. (a) ryo,,g = 0.705; P < 0.01; to,d = 0.164, NS. (b) ro,,, = 0.690, P < 0.05; to,d = 0.141, NS.
138 DISCUSSION
Our data have indicated an increase occurring with aging in the bone marrow contents of stromal precursor cells, forming the fibroblast colonies in the monolayer cultures. Such colonies, in young and old animals alike, seem to have a clonal origin, as is indirectly evidenced from the linear correlation between their number and the number of bone marrow cells being transplanted into culture. Hence the method used allowed quantification and comparison of the bone marrow CFC-F contents in mice of different age. As we have shown earlier, during the in vitro fibroblast colony formation the bone marrow cells in heterochronic associations show an additive interaction. These data as well as those on the linear relationship between the number of colonies and the number of cells being implanted in culture may suggest that it is unlikely that the increase in colony forming efficiency in old mice occurred due to activation of some stimulator cells or due to suppression of some unidentified suppressor cells. Thus, the increased colony forming efficiency of bone marrow CFC-F, probably, reflects an absolute increment of their contents in mice during aging. The assumption could be made, however, that the number of bone marrow CFCF increased during aging not at the expense of normal CFC-F, i.e., stromal cells, but rather at the expense of their progeny having limited proliferative potential. After having compared the average cellularity of fibroblast colonies in bone marrow cultures from mice of different ages, we turned down this assumption. We failed to find any significant differences in average colony cellularity between the young and old donors. The estimates thus made have shown that most colonies are formed as a result of 8--10 divisions of CFC-F (60o70 of all colonies in bone marrow cultures from young mice and 70°7o in cultures from old mice) (Fig. 5). It was concluded that with age the proliferative potential did not seem to reduce and the bone marrow CFC-F increased at the expense of the normal cells. The sensitivity of CFC-F cells to in vivo administered hydroxyurea did not show any differences in young and old mice. The stem cells - - CFC-S - - behaved in the analogous way. These results have thus showed that in aging there does not occur a marked redistribution of cells between two pools - - resting and proliferating (synthesizing the DNA) - - in the above cellular populations. Our data are at variance with the results reported by Popp, Popp (1979) demonstrating a decreased number of resting (Go) CFC-F with aging. The directly opposite results were reported by Peterson et al. (1983). The age-unrelated specifics of the behaviour of CFC-F and CFC-S following the hydroxyurea administration is worth special attention. The essence is that the bone marrow contents of CFC-F was increased, while that of CFC-S remained unchanged against a simultaneous reduction of bone marrow cellularity. Thus we have registered a peculiar phenomenon of " C F C - F concentration" due to the hydroxyurea effect (See Fig. 3). The cause of this phenomenon is unclear. A possible explanation
139
50
I-1
Young
l
40
Old f0
30-
m
0 ¢J
l
0
20o
Z
10
6
7
No.
0
of
CFC-F
II
!
11
12
divisions
Fig. 5. The histogram of distribution of the fibroblast colony numbers according to CFC-F divisions. For estimation the data of the experiments presented in Fig. 2 were us~l.
might be that these two different cell categories have different proliferative activities. Thus, according to Becker et al. [1965], only 10°70 of CFC-S proliferated in situ. Many authors, who used different methods for the assessment of proliferative activity, concluded that CFC-F did not synthetize DNA in situ [21--25]. The differences in the proliferative activity between CFC-S and CFC-F may be a cause of their dissimilar sensitivity to hydroxyurea injection. The data of the present investigation showing increased bone marrow contents of stromal precursor cells during aging are consistent with our earlier findings [11,25]. Recently, these data have been confirmed by other authors [12]. At the same time these data contradict the results obtained by Sidorovich and Latsinik (1978) who demonstrated a decrease with age in the number of clonogenic stromal precursors in bone marrow of the guinea pig. Mets and Verdonk (1981) reported a decrease in the proliferative potential of stromal colony forming cells from human bone marrow. However, no substantial changes were detected in the concentration of bone marrow CFC-F in humans [26]. According to Brockbank et al. (1983), the absolute CFC-F contents in mice did not change with aging, though the maximal age of their animals was below 17 months [10]. The most probable cause of the contradictory results might be the species-specific peculiarities of bone marrow hemopoiesis. Thus, the bone marrow cellularity in humans is reduced with age and the hemopoietic tissue is replaced by the fatty one [27]. The number of nucleated cells in the femoral bone marrow of the 1-year-old guinea pig does not differ essentially from that of the 2-
140
month-old animal [8]. In mice, as evidenced by numerous observations, the cellularity of femoral bone marrow is almost doubled during aging (See Data Review 17). As shown in this study, apart from the increase in the absolute number of hemopoietic cells in old mice there is also the increase in the bone marrow contents of the stromal precursor cells. We have also established a direct linear relationship between the number of CFC-F and the number of nucleated cells or CFC-C in young mice. The similar pattern of the relationship between CFC-F and CFC-C was found in humans [28]. According to the data of Jinnai et al. (1984), the number of CFC-F correlates positively with the number of erythroid (CFC-E and BFC-E) but not granulocytic-macrophagal (GM-CFC) precursors from human bone marrow. Piersma et al. (1983) also pointed to the existence of positive correlation between the bone marrow contents of CFC-F and the regenerative potential of subcutaneous implants of the femoral bones. The presence of the relationship between bone marrow CFC-F contents and various categories of hemopoietic cells in mice may suggest that CFC-F are involved in the construction of-stromal tissue microenvironment and that its organisation with age may be reflected in altered (increased) CFC-F in bone marrow of mice. Probably, this reorganisation is aimed to provide "preserving" of stromal hemopoietic cells and even increasing the total hemopoietic tissue mass of the femoral bone marrow in old mice. Meanwhile, the direct correlation between the CFC-F number and the total number of hemopoietic and GM-CFC cells are altered during aging. Interestingly, the direct correlation between CFC-F and GM-CFC seen in healthy human subjects is lost during hematologic diseases [22,28]. If taken together, these data may indicate that in hemopoiesis pathology as well as during aging the stromal tissue ceases to perform its microenvironmental functions. It is not excluded however that both in pathology and during aging the changes in hemopoietic and stromal precursor cell contents and the changes in the microenvironmental function of the stroma occur asynchronically. The succession and the interrelation of these processes are unclear and deserve a thorough investigation. Thus, the results of the present investigation have shown that the femoral bone marrow contents of stromal precursor cells for fibroblast colonies are increased in old mice. CFC-F from the bone marrow of old animals do not differ as regards their proliferative potential and proliferative activity from C F C - F from young animals. So far, no conclusion can be made whether the microenvironmental functions of the stromal tissue undergo a change during aging, though the absence of correlation between the bone marrow contents of C F C - F and the hemopoietic cells might evidence indirectly for such a possibility. ACKNOWLEDGEMENT
The authors wish to acknowledge Maya Tourta for her assistance in preparing the manuscript as well as to thank the referees for their suggestions and editing our manuscript.
141 REFERENCES 1 2 3 4 5 6
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11 12 13 14 15 16
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A.V. Sidorenko, I.B. Gubrii, L.F. Andrianova, T.V. Macsijuk and G.M. Butenko, Functional rearrangement of lymphohemopoietic system in heterochronically parabiosed mice. Mech. Ageing Dee., 36 (1986) 41--56. 26 D. Zipori, N. Reichman, L. Arcavi, M. Shtalrid, A. Berrebi and P. Resnitzky, In vitro functions of stromal cells from human and mouse bone marrow. Exp. Hematol., 13 (1985) 603--609. 27 R.J. Hartsock, E.B. Smith and C.S. Petty, Normal variation with aging on the amount of hvmatopoietic tissue in bone marrow from the anterior iliac crest. Am. J. Clin. PathoL, 43 (1965) 326--331. 28 G.L.B. Scarra, R. Barresi, F. Ajmar, C. Gaffe, R. Ravazzolo and M. Sessarego, Fibroblast colonyforming cells in myeloproliferative disorders. Acta HaematoL, 70 (1983) 349--356. 29 J. Jinnal, M. Bessho, I. Murohashi, N. Nara and K. Hirashima, Rdationship between fibroblastoid colony-forming units (CFU-F) and hemopoietic precursor cells in normal human bone marrow. Int. J. Cell Cloning, 2 (1984) 341--347. 30 A.H. Piersma, R.E. Ploemacher and K.G.M. Brockbank, Radiation damage to femoral hemopoietic stroma measured by implant regeneration and quantitation of fibroblastic progenitors. Exp. HematoL, H (1983) 884--890.
131
ElsevierScientificPublishers Ireland Ltd.
STROMAL H E M O P O I E T I C M I C R O E N V I R O N M E N T IN A G I N G
A.V. SIDORENKO, BUTENKO
L.F. ANDRIANOVA*,
T.V. MACSYUK AND G.M.
Laboratory of Pathophysiology, Institute of Gerontology of the Academy of Medical Sciences of the U.S.S.R., Vyshgorodskaya St. 67, 252655, Kiev 114 (U.S.S.R.)
(ReceivedAugust 29th, 1989) (RevisionreceivedDecember I st, 1989)
SUMMARY The bone marrow content, proliferative potential and proliferative activity of precursor cells for stromal fibroblast colony forming cells (CFC-F) were investigated in young and old CBA mice. The relationship between CFC-F and the number of bone marrow nucleated cells and granulocytic-macrophagal precursors (GM-CFC) was studied as well. The results obtained showed increased CFC-F contents in old animals. The proliferative potential of old mice CFC-F did not appear to differ from that of young animals. The proliferative activity of bone marrow CFC-F and hemopoietic stem cells - - spleen colony forming cells (CFC-S) was studied by determining the sensitivity to hydroxyurea administration. The responses were almost the same in young and old mice. A direct correlation between CFC-F and nucleated cells and GM-CFC precursors was found in young mice, but not in the old animals. The results of the present study have pointed to the reorganisation o f the stromal tissue microenvironment in bone marrow in old age.
K e y words: Bone marrow; Hemopoietic and stromal precursor cells; Aging
INTRODUCTION Age-related variations o f the lymphohemopoietic system, especially of its lymphoid part, are well documented [1--3]. Nevertheless, the mechanisms of these changes remain unclear. In the most general sense the problem is to distinguish the extrinsic (macro-, microenvironment) and intrinsic (mainly, genetic program) factors, modulating the hemopoietic and lymphoid cell functions during aging. *To whom all correspondenceshould be addressed. 0047-6374/90/$03.50 Printed and Published in Ireland
© 1990ElsevierScientificPublishers Ireland Ltd.
132 The stromal tissue seems to be one of the most important sources of extrinsic signals, which can cause the age-dependent changes in lymphohemopoiesis [4--6]. In this connection it was interesting to assess the functional organisation and activity of the lymphohemopoietic stromal microenvironment in aging. For this purpose, the experimental approach that involved the quantitation of stromal precursor cells (CFC-F) was used. The above cells provided clonal growth of fibroblast colonies in primary monolayer cultures o f lymphoid and hemopoietic tissues [7]. CFC-F occupy the central place in a stromal cell hierarchy responsible for transfer of the hemopoietic microenvironment and serve as "stromal stem cells" [7]. The experimental attempts to evaluate the CFC-F contents in bone marrow of young and old laboratory rodents and humans have yielded contradictory results [8--12]. In the present study we have compared the contents and some proliferative characteristics of the femoral CFC-F in young and old CBA mice. MATERIALSAND METHODS
Animals Female CBA mice (Stolbovaya Breeding Laboratory, U.S.S.R. Academy o f Medical Sciences) were used when 2--3 months (young) and 22--26 months (old) of age. Only healthy appearing mice found to be grossly free o f disease at autopsy were evaluated. Outbred guinea pigs of both sexes were used as a source o f bone marrow feeder cells in CFC-F cloning experiments.
Preparation of cell suspensions Femurs were removed aseptically, epiphyses were removed and bone marrow was flushed into ice-cold RPMI-1640 media (Flow Laboratories, Glasgow, Scotland). To prepare single cell suspension cells were passed repeatedly through syringe with needles o f various diameters. The obtained suspensions were employed for count of nucleated cells and for cloning hemopoietic and stromal precursor cells.
Hemopoietic cells (1) Femoral bone marrow nucleated cells were calculated in a hemocytometer using 3 °7o acetic acid solution. (2) Hemopoietic stem cells (CFC-S) were determined according to their ability to form colonies in spleens of lethally irradiated mice [13]. Young (3 months) irradiated (10 Gy) recipient mice were injected with 0.75 x 105 bone marrow cells from young or old donors. The recipients were killed at day 8 after grafting and the number of macroscopic hemopoietic colonies corresponding to the number of CFC-S was counted on the spleen surface. (3) The granulocyte-macrophage colony-forming cells (GM-CFC) were estimated
133 using the agar cultures of bone marrow cells [14]. Medium conditioned by spleen cells incubated for 72 h in the presence o f 5 mkg/ml Concanavalin A (Calbiochem, San Diego, CA) was used as a source o f colony-stimulating factor (CSF) [15]. 105 bone marrow cells were cultivated in supplemented McCoy's 5A medium (Serva, Heidelberg, F.R.G.) until day 8 when the microscopic colonies growing in cultures were scored [16].
Fibroblast colony-forming cells Femoral bone marrow was assayed for the content o f fibroblast colony-forming cells using the method described in detail elsewhere [16]. Briefly, murine bone marrow cells at starting plating density of 2.5 X 105 cells/cm 2 were cultivated along with irradiated (45 Gy) guinea pig bone marrow cells (3.5 X 105 cells/cm z) in 85070 RPMI 1640 medium with 15 070 o f fetal calf serum (Flow Laboratories, Glasgow, Scotland). The cultures were incubated for 12 days at 37°C in a humidified atmosphere of 10070 CO 2 and 9007o o f room air. At the end o f incubation, the cultures were fixed with 96°70 ethanol, stained with azur-eosin and examined under dissection microscope to score colonies consisting of at least 50 fibroblast cells.
Proliferative activity of CFC-F and CFC-S Hydroxyurea (Serva, Heidelberg, F.R.G.) that can eliminate selectively cells in Sphase of the cellular cycle was injected intraperitoneally in the dose of 1 mg/kg of body weight twice with a 7-h interval to the young and old mice. Sixteen to eighteen hours following the last injection the numbers of nucleated cells, CFC-F and CFC-S were determined in the femoral bone marrow o f the animals.
Statistics Student's t-test was used to evaluate all the data. RESULTS As seen in Table I, the efficiency of stromal fibroblast colony formation by bone marrow cells in the old mice was almost two times higher compared to the young animals. The absolute number of CFC-F in bone marrow from old animals was also increased due to significantly increased nucleated cell numbers in the bone marrow during aging (Table I). There were similar observations during explantation into culture of cells from individual donors or when bone marrow ceils from several donors were pooled. It should be stressed that there were no significant differences in average CFC-F values in age-matched animals when the above two approaches were applied. The linear relationship was found between the number of bone marrow cells being explanted into culture and the number o f grown fibroblast colonies (Fig. 1). Such a relationship was characteristic o f both young and old cells.
134
TABLE I THE NUMBER OF N U C L E A T E D CELLS A N D CFC-F IN BONE MARROW FROM YOUNG AND OLD MICE
Age
Method o f testing
No. o f experiments
No. o f nucleated cells in the f e m u r ( X IO5)
No. o f CFC-F in explanted cells, 10~
No. o f CFC-F in bone marrow o f the femur
Young
Inand individual mouse InapooP Inan individual mouse Inapool
11
131.45 ±
2.24 -+ 0.23
302.27 + 36.76
16 16
141.13 ± 10.64 200.38 ± 11.84"**
2.48 ± 0.31 4.40 - 0.46**
326.00:1:36.01 808.94 ± 87.10"**
15
201.73 ± 17.38"*
3.63 ± 0.30*
704.13 :l: 61.04"**
Old
7.37
The differences of corresponding indices between young and old mice are statistically significant: *P < 0.05; * * P < 0.01; and * * * P < 0.001. aTwo or three mice were used in each experiment if bone marrow cells were pooled.
For comparison of the proliferative potential of CFC-F in bone marrow of the young and old mice, the experiments were set up as paired (young/old) observations. The average cellularity of colonies was found to be almost the same in the young and old animals (Fig. 2).
200-
0~~
o u
100.
Jo
0
u. o
z
0
NO. OF
CELLS
P L A T E D ('106)
Fig. I. The linear relationship between the number of fibroblast colonies and the number of explanted bone marrow cells for young (O - - - O; r = 0.906; y = 2.92x + 42.78; P < 0.01 and old ( e - - e ; r = 0.948; y = 3.19x + 39.13) mice. The data are presented as the means of triplicate cultures from three different experiments.
135
A N3
O >Z O
g,
2-
U
u.I D.
| o•
1 0
g
m
u.. 0
6 Z
young
old
Fig. 2. Fibroblast colony cellularity in bone marrow cultures from young and old mice. At the end of incubation (at day 12), the cultures were fixed and stained and the number of fibroblasts in the grown colonies was counted, using a dissection microscope. At least 52 colonies from young and 50 colonies from old animals were used. Mean cellularity values were 532 ± 46 and 566 ± 58, respectively.
TABLE II THE EFFECT OF IN VITRO A D M I N I S T E R E D H Y D R O X Y U R E A ON THE NUMBER OF NUCLEATED CELLS, CFC-F A N D CFC-S IN BONE MARROW OF YOUNG AND OLD MICE
Age
Hydroxyurea administration
No. o f nucleated cells in the f e m u r ( x 10~)
No. o f CFC-F: In explanted cells, 10~
In bone marrow In bone o f the f e m u r marrow 10~cells
In bone marrow o f the femur
Young
--
119 _ 13
2.30 __ 0.17
309 ± 29
17.0 __ 1.4
1615 _+ 356
Old
+ -+
47 ± 205 ± 94 ±
3.90 ± 0.36*** 3.90 __ 0.16 4.70 ± 0.20**
152 -+ 13"** 775 ± 25 389 ± 24***
16.3 _+ 1.6 12.7 "4" l.O 13.0 _+ 4.7
735 ± 128" 2564 - 167 1146 ± 429**
9*** 6*** 6***
No. o f CFC-S:
The results are mean values of bone marrow cellularity and CFC-S contents for 5 experiments and of CFC-F cloning for three experiments. The differences of indices between control and experimental (hydroxyurea treatment) animals of the same age are signficant: *P < 0.05; **P < 0.01; and ***P < 0.001.
136
To evaluate the proliferative activity, the parallel study was undertaken on the effect of in vitro administration of hydroxyurea on stromal (CFC-F) and hemopoietic (CFC-S) precursor cells from bone marrow of young and old mice. The data of these experiments are presented in Table II. After hydroxyurea injection, the fibroblast colony forming efficiency o f bone marrow cells increased in cultures from young and old animals alike (Fig. 3). At the same time, the hemopoietic colony forming efficiency did not change significantly in either animal group (See Table II). Because o f the hydroxyurea-induced decrease in the total nucleated cell numbers of the bone marrow, its contents o f the precursor cells, both stromal and hemopoietic, decreased considerably. It is noteworthy that bone marrow cellularity and its CFC-F and CFC-S contents in young and old mice reduced in about the same manner. For example, the number of nucleated cells in femoral bone marrow decreased to 40 _+ 8070 in young and 46 _.+ 307o in old animals (P > 0.05). The number of CFC-S decreased to 38 ___ 9070 and 45 _.+ 6070 (P > 0.05), respectively, while the number of CFC-F decreased to 49 _+ 4070and 50 _.+ 3070 ( P > 0.05), respectively. We then tried to find out if there existed a correlation between the bone marrow contents of CFC-F and hemopoietic cells - - nucleated cells and GM-CFC. Again, we found a substantial increase with age in the number of nucleated cells and CFC-F (Table III). As regards GM-CFC, we failed to detect any meaningful character of increase in the precursor cells in old animals, probably, because of the varying
$
4
Fig. 3. The number of fibroblasts from bone marrows of young (1,2) and old (3,4) mice, control (1,3) and in vivo administered hydroxyurea (2,4).
137 T A B L E III T H E RESULTS O F T H E P A R A L L E L ASSESSMENT OF T H E N U M B E R OF H E M O P O I E T I C A N D S T R O M A L P R E C U R S O R C E L L S IN T H E F E M O R A L BONE M A R R O W F R O M Y O U N G A N D O L D MICE
Indices
Young mice (n = 12)
No. o f the nucleated cells (105) No. o f G M - C F C per 105 o f the explanted cells No. of G M - C F C in whole bone marrow o f the femur No. o f C F C - F per 10~ o f the explanted cells No. o f CFC-F in whole bone marrow of the femur
mice 14)
Old
(n
=
189 3: 13'** 103 3 : 1 9
124 3 : 1 0 99 3 : 1 6 14 399 3:2849
20 333 3:4156
2.48 3:0.28
4.55 3: 0.58***
3 I0 3 : 4 3
811 _+ 103
The differences of corresponding indices between y o u n g and old mice are statistically significant: ***P < 0.001.
cloning efficiency from one experiment to another. The number of CFC-F appeared to correlate positively both with the total number of hemopoietic (nucleated) cells, and with the number of GM-CFC in the young mice but not in the old animals (Fig. 4). o
"7.
/
5-
t_
/
E
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•
co
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/ °°
~
E
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ez •
:/
u~ oJ u
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~'
•
•
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,~.
3
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2
o
•
/
0
6
u,. (J
0
1
i.i Z
0
0
I 2
,~
I
I 81'01
12
l 14
CFC-F
0
I
2 per
femur
(.lO
I
4
I
6
I
8
110
112
I
14
2)
Fig. 4. The relationships between C F C - F and the total n u m b e r of nucleated cells (a) or granulocyte/ macrophage colonies per femur (b) in y o u n g (O - - O) and old ( e - - e ) mice. (a) ryo,,g = 0.705; P < 0.01; to,d = 0.164, NS. (b) ro,,, = 0.690, P < 0.05; to,d = 0.141, NS.
138 DISCUSSION
Our data have indicated an increase occurring with aging in the bone marrow contents of stromal precursor cells, forming the fibroblast colonies in the monolayer cultures. Such colonies, in young and old animals alike, seem to have a clonal origin, as is indirectly evidenced from the linear correlation between their number and the number of bone marrow cells being transplanted into culture. Hence the method used allowed quantification and comparison of the bone marrow CFC-F contents in mice of different age. As we have shown earlier, during the in vitro fibroblast colony formation the bone marrow cells in heterochronic associations show an additive interaction. These data as well as those on the linear relationship between the number of colonies and the number of cells being implanted in culture may suggest that it is unlikely that the increase in colony forming efficiency in old mice occurred due to activation of some stimulator cells or due to suppression of some unidentified suppressor cells. Thus, the increased colony forming efficiency of bone marrow CFC-F, probably, reflects an absolute increment of their contents in mice during aging. The assumption could be made, however, that the number of bone marrow CFCF increased during aging not at the expense of normal CFC-F, i.e., stromal cells, but rather at the expense of their progeny having limited proliferative potential. After having compared the average cellularity of fibroblast colonies in bone marrow cultures from mice of different ages, we turned down this assumption. We failed to find any significant differences in average colony cellularity between the young and old donors. The estimates thus made have shown that most colonies are formed as a result of 8--10 divisions of CFC-F (60o70 of all colonies in bone marrow cultures from young mice and 70°7o in cultures from old mice) (Fig. 5). It was concluded that with age the proliferative potential did not seem to reduce and the bone marrow CFC-F increased at the expense of the normal cells. The sensitivity of CFC-F cells to in vivo administered hydroxyurea did not show any differences in young and old mice. The stem cells - - CFC-S - - behaved in the analogous way. These results have thus showed that in aging there does not occur a marked redistribution of cells between two pools - - resting and proliferating (synthesizing the DNA) - - in the above cellular populations. Our data are at variance with the results reported by Popp, Popp (1979) demonstrating a decreased number of resting (Go) CFC-F with aging. The directly opposite results were reported by Peterson et al. (1983). The age-unrelated specifics of the behaviour of CFC-F and CFC-S following the hydroxyurea administration is worth special attention. The essence is that the bone marrow contents of CFC-F was increased, while that of CFC-S remained unchanged against a simultaneous reduction of bone marrow cellularity. Thus we have registered a peculiar phenomenon of " C F C - F concentration" due to the hydroxyurea effect (See Fig. 3). The cause of this phenomenon is unclear. A possible explanation
139
50
I-1
Young
l
40
Old f0
30-
m
0 ¢J
l
0
20o
Z
10
6
7
No.
0
of
CFC-F
II
!
11
12
divisions
Fig. 5. The histogram of distribution of the fibroblast colony numbers according to CFC-F divisions. For estimation the data of the experiments presented in Fig. 2 were us~l.
might be that these two different cell categories have different proliferative activities. Thus, according to Becker et al. [1965], only 10°70 of CFC-S proliferated in situ. Many authors, who used different methods for the assessment of proliferative activity, concluded that CFC-F did not synthetize DNA in situ [21--25]. The differences in the proliferative activity between CFC-S and CFC-F may be a cause of their dissimilar sensitivity to hydroxyurea injection. The data of the present investigation showing increased bone marrow contents of stromal precursor cells during aging are consistent with our earlier findings [11,25]. Recently, these data have been confirmed by other authors [12]. At the same time these data contradict the results obtained by Sidorovich and Latsinik (1978) who demonstrated a decrease with age in the number of clonogenic stromal precursors in bone marrow of the guinea pig. Mets and Verdonk (1981) reported a decrease in the proliferative potential of stromal colony forming cells from human bone marrow. However, no substantial changes were detected in the concentration of bone marrow CFC-F in humans [26]. According to Brockbank et al. (1983), the absolute CFC-F contents in mice did not change with aging, though the maximal age of their animals was below 17 months [10]. The most probable cause of the contradictory results might be the species-specific peculiarities of bone marrow hemopoiesis. Thus, the bone marrow cellularity in humans is reduced with age and the hemopoietic tissue is replaced by the fatty one [27]. The number of nucleated cells in the femoral bone marrow of the 1-year-old guinea pig does not differ essentially from that of the 2-
140
month-old animal [8]. In mice, as evidenced by numerous observations, the cellularity of femoral bone marrow is almost doubled during aging (See Data Review 17). As shown in this study, apart from the increase in the absolute number of hemopoietic cells in old mice there is also the increase in the bone marrow contents of the stromal precursor cells. We have also established a direct linear relationship between the number of CFC-F and the number of nucleated cells or CFC-C in young mice. The similar pattern of the relationship between CFC-F and CFC-C was found in humans [28]. According to the data of Jinnai et al. (1984), the number of CFC-F correlates positively with the number of erythroid (CFC-E and BFC-E) but not granulocytic-macrophagal (GM-CFC) precursors from human bone marrow. Piersma et al. (1983) also pointed to the existence of positive correlation between the bone marrow contents of CFC-F and the regenerative potential of subcutaneous implants of the femoral bones. The presence of the relationship between bone marrow CFC-F contents and various categories of hemopoietic cells in mice may suggest that CFC-F are involved in the construction of-stromal tissue microenvironment and that its organisation with age may be reflected in altered (increased) CFC-F in bone marrow of mice. Probably, this reorganisation is aimed to provide "preserving" of stromal hemopoietic cells and even increasing the total hemopoietic tissue mass of the femoral bone marrow in old mice. Meanwhile, the direct correlation between the CFC-F number and the total number of hemopoietic and GM-CFC cells are altered during aging. Interestingly, the direct correlation between CFC-F and GM-CFC seen in healthy human subjects is lost during hematologic diseases [22,28]. If taken together, these data may indicate that in hemopoiesis pathology as well as during aging the stromal tissue ceases to perform its microenvironmental functions. It is not excluded however that both in pathology and during aging the changes in hemopoietic and stromal precursor cell contents and the changes in the microenvironmental function of the stroma occur asynchronically. The succession and the interrelation of these processes are unclear and deserve a thorough investigation. Thus, the results of the present investigation have shown that the femoral bone marrow contents of stromal precursor cells for fibroblast colonies are increased in old mice. CFC-F from the bone marrow of old animals do not differ as regards their proliferative potential and proliferative activity from C F C - F from young animals. So far, no conclusion can be made whether the microenvironmental functions of the stromal tissue undergo a change during aging, though the absence of correlation between the bone marrow contents of C F C - F and the hemopoietic cells might evidence indirectly for such a possibility. ACKNOWLEDGEMENT
The authors wish to acknowledge Maya Tourta for her assistance in preparing the manuscript as well as to thank the referees for their suggestions and editing our manuscript.
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