Mechanisms of Ageing and Deveiopment, 56 (1990)89--97
89
ElsevierScientificPublishers Ireland Ltd.
AGE-RELATED DECREASE IN THE NUMBER OF HEMOPOIETIC STEM CELLS AND P R O G E N I T O R S IN SENESCENCE A C C E L E R A T E D MICE
HIROKO IZUMI-HISHA"*, YOSHITAKE ITO b, KENKICHI SUGIMOTO ~, H I D E H I K O OSHIMA" and K A Z U H I R O J. MORF "Department of Hygiene, Aichi Medical University and blnstitute for Aging Research, Aichi Medical University, Aichi and "Department of Biology, Faculty of Science, Niigata University, Niigata (Japan)
(ReceivedDecember14th, 1989) (Revision received June 8th, 1990)
SUMMARY Senescence accelerated mice (SAM-P) were used for the study o f the possible aging of hemopoiesis. The number of peripheral leukocytes decreased significantly with age, whereas hematocrit showed only a slight decrease. Although the number o f total nucleated cells in the bone marrow increased, the number of hemopoietic stem cells (CFU-S) as well as that of granulocyte-macrophage colony forming cells (GM-CFC) showed a decrease in old mice. A significant decrease in the number of GM-CFC was observed in the spleen o f old SAM-P mice, whereas no decrease was found in the number of CFU-S. Such a profound reduction o f the recruitment of GM-CFC from CFU-S in the spleen together with a reduction of bone marrow hemopoiesis may be responsible for the decrease in the number of peripheral leukocytes in the old mice. SAM-P mice could provide a good model for the study of the aging of hemopoietic system. K e y words: Aging; Hemopoiesis; Senescence accelerated mice; Hemopoietic stem
cells; Granulocyte macrophage progenitor cells INTRODUCTION Pluripotent hemopoietic stem cells (CFU-S) have a capacity of extensive selfrenewal. Aging of hemopoiesis, especially o f self-renewal capacity of CFU-S has been a matter o f debate for a long time. *Present address and to whom all correspondence should be addressed: First Department of Pathology, Kansai Medical University,Fumizono-cho1, Moriguchi-shi,Osaka, 570, Japan.
0047-6374/90/$03.50 Printed and Published in Ireland
© 1990ElsevierScientificPublishers Ireland Ltd.
90
A number of investigators reported that the self-renewal capacity as well as the number of CFU-S remained unchanged during the life span [1] although the hematocrit [2] and the population size of granulocyte-macrophage progenitor cells (GMCFC) in cycle [3] decreased with age. The repeated transplantation of hemopoietic stem cells into lethally-irradiated recipient mice resulted eventually in a loss of the ability to reconstitute hemopoietic system in host mice within 6 - - 8 serial transplantation [4]. lnoue and Cronkite [5] reported that the hemopoietic micro-environment of old mice had a reduced capacity to support the spleen colony formation derived from CFU-S with regard to the size of the spleen colonies. These contradictory findings might be due to the difference in the experimental procedures. Takeda et al. [6,71 established S A M - P / I - P / 5 mice (Senescence prone series of Senescence Accelerated Mouse), which have a short life span of about 9 months, from A K R / J mice. These mice were found to have an accelerated deposition of amyloid in their lung, spleen, liver, kidney etc., except brain and bone, after the age of 24 weeks [8--10]. Other characteristics of aging, such as cataract, periophthalmic lesion [11], increased skin coarseness, alopecia, increased lordokyphosis of spine and so on, were also observed. Such a manifestation of aging appears after normal growth, that is after 20--25 weeks and their aging advances in a accelerated manner. SAM-P mice would, therefore, be a suitable model of the study of the possible aging of the hemopoietic system. As controls, senescence resistance series (SAM R / I-R/3) were also established from A K R / J mice. Their life span is 13.3 months and their aging pattern is similar to other normal strains, when compared with that of SAM-P. The present study describes hemopoietic features of SAM-P mice in order to obtain fundamental data of age-related hemopoietic changes. MATERIALS AND M E T H O D S
Animals
A K R / J strain mice were originally donated from The Jackson Laboratory (Bar H a r b o r , ME). Senescence-prone series (SAM-P) were obtained by serial mating of the litters which had a short life span, by Takeda et al. [6,7]. Senescence-resistant series (SAM-R) were also established in 1981. These mice were reared under near specific pathogen-free (SPF) conditions and fed with a commercial diet (CE-2, Nihon Clea) and tap water ad libitum. The mice were used at the age of 8 weeks or 33 weeks, because the mice of these series have been well examined. Male SPF mice of BDF~ strain were purchased from the Charles River Breeding Laboratories (Japan) at the age of 8 weeks and maintained under SPF conditions. The mean life span of this strain is approximately 30 months at our animal facilities. Male SPF mice of ddY strain were obtained from the Shizuoka Laboratory Animal Center (Japan) and used at the age of 8 weeks or 33 weeks.
91
Peripheral blood Blood was obtained by cardiac or retro-orbital sinus puncture under ether anaesthesia and the number o f white blood cells (WBC) was counted for each mouse individually. Hematocrit (Hct) was also measured for each mouse.
Cell preparations Bone marrow and spleen cells were collected and pooled from 8 to I0 mice per each point. Single cell suspension were prepared and used for both CFU-S and GMCFC assays. In each experiment, the cells from young and old mice of the same strain were assayed simultaneously.
GM-CFC assay Number of GM-CFC in the bone marrow and the spleen was measured by the soft-agar culture technique using a modified method of Bradley and Metcalf [12]. Briefly, 1--2 x l05 bone marrow cells or 2 - - 4 × 106 spleen cells were plated into petri dishes containing 2 ml of Fischer's medium supplemented with 0.3070 agar, 25°70 horse serum and 10070 mouse lung-heart conditioned medium as colony-stimulating factor (CSF). A minimum of three replicate dishes were cultured for each point. The dishes were incubated for 7 days and the number of cell clusters consisted o f more than 50 cells were counted as colonies.
CFU-S assay Aliquots of the bone marrow or spleen cell suspension were used for the assay of CFU-S according to the method of Till and McCulloch [13]. For SAM-P mice, the appropriate number of bone marrow or spleen ceils from 6 to 10 mice were injected intravenously into 8-week-old SAM-R mice (10--13 per point) that had been irradiated with 8.0 Gy X-rays. The cells from other strains were injected i.v. into lethally irradiated (8.0 Gy for SAM-R, 9.5 Gy for BDF I and 8.0 Gy for ddY) mice. The recipient mice were sacrificed 10 days after the cell-injection and their spleens were fixed in Bouin's solution. The nodules on the surface of the spleen were counted after 1 day of fixation. RESULTS
Hemopoietic features o f SA M-P and SA M-R mice (a) Peripheral blood. The change in the values of WBC and Hct with age was investigated in both SAM-P and -R mice. Results are summarized in Table I. There was no difference either in WBC count or in Hct at the age of 8 and 16 weeks. The number of WBC at 33 weeks, however, reduced to half of that at 8 weeks in SAM-P mice (significant, P < 0.01). A slight decrease in the number of WBC was observed in SAM-R. The value of Hct decreased slightly at the age of 33 weeks only in SAM-P mice.
92
TABLE I A G E - A S S O C I A T E D C H A N G E S IN T H E F E A T U R E S O F P E R I P H E R A L
B L O O D IN S A M M I C E
8 Weeks
16 Weeks
33 Weeks
Whiteblood cells(/lal)
SAM-P SAM-R
6900 __. 1000 7200 :i: 800
7700 :t: 1000 7200 __. 1300
3500 :t: 200* 5700 _+ 700
Hematocrit (%)
SAM-P SAM-R
44 -*- 0.8 44 --- 0.8
43 :t: 1.0 43 --. 0.7
41.5 _ 1.5 45 --. 2.0
The values represent the m e a n ± S.E. o f 8 - - 1 0 mice. *Significant c h a n g e f r o m 8 weeks ( P < 0.01).
(b) Bone marrow andspleen. The change in the number of hemopoietic cells with age was also examined for bone marrow and spleen. The number of CFU-S and GMCFC in old SAM-P mice (33 week-old) expressed as the number per 105 cells were about 40070 of the number at 8 week-old mouse bone marrow (significant P < 0.01). As the number of total nucleated cells per femur increased with age, the net decrease of the stem cell population was about 30070 . A similar decrease in the number of GM-CFC was observed in SAM-R mice at 33 weeks (P < 0.01). No decrease was observed, however, in the number of CFU-S in these mice (Table II (a)). A marked decrease in the number of GM-CFC was observed in the spleen of both SAM-P and-R mice at 33 weeks (P < 0.01). The number of CFU-S, however, did not show any significant decrease in either of these mice (Table II (b)). The fact that a significant decrease with age of the number of GM-CFC occurs in SAM-R mice, makes it unsuitable to use SAM-R mice as 'normal' controls of SAMP mice. Effect of age on hemopoiesis o f normal mice (a) Peripheral blood. BDF~ and ddY mice were used as normal control and the changes in WBC count and Hct value were determined at various ages (Table III). No significant changes in the number of WBC or Hct values were observed in ddY mice at the age of 33 weeks. In BDF~ mice, Hct showed only slight decrease (insignificant). The number of WBC showed a slight increase at the age of 2 years compared to the number at the age of 8 weeks (insignificant). (b) Bone marrow andspleen. Numbers of GM-CFC and CFU-S of old mice were compared with those of 8 or 9 weeks. The ratio of 33 weeks:8 or 9 weeks is shown in Fig. 1. In order to facilitate the comparison with SAM-P mice, their results are also added to the figure. In ddY mice, numbers of GM-CFC and CFU-S per 105 bone marrow ceils at 33 weeks were 70--80°70 of the numbers of young mice. Similar decrease was also observed in BDF t mice at the age of I year + 10 weeks. The degree of the reduction, however, was smaller than that in SAM-P mice (insignificant). Since the number of
93
TABLE II A G E - A S S O C I A T E D C H A N G E S IN T H E NUMBER OF H E M O P O I E T I C C E L L P O P U L A T I O N IN BONE M A R R O W A N D S P L E E N OF SAM M I C E
(a) Bone marrow (1) SAM-P mice Nucleated cells CFU-S GM-CFC
/10~
/femur /10~
/femur (2) SAM-R mice Nucleated cells
CFU-S GM-CPC
/10~
/femur /los
/femur (b) Spleen (1) SAM-P mice Nueleatedcells CFU-S GM-CFC
/los
/spleen /los
/spleen (2) SAM-R mice
Nucleated cells /los CFU-S
/spleen
GM-CFC
/106
/spleen
8 Weeks
33 Weeks
Ratio o f 33 wk/8 wk
8.6 X los 25 ± 4 2150 ± 310 266 ± 25 22 876 ± 2167
17.8 x 9 1600 103 18 300
los ± 4" ± 730 ± 19" ± 3376
2.1 0.4 0.7 0.4 0.8
8.9 x los 29 ± 6 2554 ± 561 166 ± 5 14774 ± 445
14.4 x 27 3900 85 12200
106 ± 6 ± 821 ± 8* ± 1080
i.6 0.9 1.5 0.5 0.8
2.1 x los 58 ± 6 12243 ± 1323 200 ± 20 42 000 ± 4288
2.8 × 57 15 390 53 14 800
l 0s ± 5 ± 1700 ± 9* ± 2380
1.3 1.0 1.3 0.3 0.4
2.2 × los 21 ± 4 4620 ± 836 83 ± 15 18 260 ± 3380
3.1 × 20 8100 26 5700
los ± 6 ± 1562 ± 3* ± 992
1.4 1.4 1.8 0.3 0.3
The values represent the mean ± S.E. of 8 - - 10 mice. *Significant change from 8 weeks ( P < 0.01).
total bone marrow cells increased significantly with age, the total numbers o f GMCFC and CFU-S in bone marrow showed an increase at the age o f 1 year and 1 year + 10 weeks o f BDF. mice. This contrasts the marked decrease o f the stem cell populations at the age o f 33 weeks in SAM-P mice. N o significant decrease was observed in the number o f GM-CFC or CFU-S per 105 cells in the spleen o f ddY mice at the age o f 33 weeks (insignificant). Old mice of BDF~ strain (2 years old) showed a slight decrease in the number o f GM-CFC in the spleen, but it was not significant. DISCUSSION
In the first part o f this study, we examined the age-related change in hemopoiesis
94
!!-
(1) Bone ~
0
C4dle 8AM-P/1
ddY
(33vd~
BOF!
(33v4~)
(11mr)
( 11mar+lO vb.)
(2 yoars)
-
# t o t l d CleU.8 GM-(:PC
tOtM (]qJ-6 GM4:FC
total CF'J-8 OM.O:C: t,vld CFU-S GM-CFC
totad O:U-S
(2] ~:ie4m Ceils SM4-P/1
]
~
I
8DF~
r
,--,
8
0
~ tOial (]:U.4 G M . O ~ SC
total (]=U-S GM.O:C SC
: TOlal nuckmled c44t$ io~r fen~r ow Wieen
total CFQ-S GM.CFC SC
Fig. 1. Change in the number of hemopoietic cell population in bone marrow and spleen. Bone marrow and spleen cells were collected from mice of various ages and assayed for the number of GM-CFC and CFU-S. The results are expressed as percentage of the value at 8 weeks.
o f S A M - P and -R mice. No significant difference in the aging o f hemopoiesis was observed between these two groups o f mice. Deposition o f amyloid and other characteristics o f aging are also observed in S A M - R mice and therefore it seems that S A M - R mice finally shows aging o f the same substance as S A M - P mice. However, the time o f appearance o f aging and its degree in S A M - R mice are different f r o m those in S A M - P mice. It is shown that genetic profiles o f both mice are already different (unpublished data). It seems that their separation is not perfect yet as to hemopoiesis. Accordingly, we used old mice o f normal strains (ddY and BDF~) as a control o f S A M - P mice, because it is well-known that the aging pattern and the hemopoitic characteristics are n o r m a l in both strains. A significant decrease in the n u m b e r o f W B C was observed at 33 weeks o f S A M - P mice, whereas such an obvious decrease did not occur in these n o r m a l mice (Tables I, III). N u m b e r s o f C F U - S and G M - C F C per l0 s b o n e m a r r o w cells at the age o f 33 weeks o f S A M - P mice reduced to 40°70 o f those at 8 weeks (Fig. l-(l)). The total n u m b e r o f these cells also reduced as a result, although the total cellularity o f the bone m a r r o w increased with age. In n o r m a l mice, on the other hand, the decrease o f the stem cells and progenitor cells was small and even an increase o f the n u m b e r o f CFU-S and G M - C F C was observed at 1 year in BDF~ mice. Aging o f hemopoietic system is, if any, ascribed either to stem cells themselves [4]
43 ± 3.0
*The value represents the mean ± S.E. **Number of mice used.
(6)
Hematocrit
(~)
6700 -4- 600* (5)**
Whiteblood cells (/pl)
(7)
43 + 0.9
6300 ± 700 (7)
(8)
39 + 1.2
5800 ± 500 (9)
9 Weeks
8 Weeks
33 Weeks
BDFI
ddY
CHANGE IN THE FEATURES OF P E R I P H E R A L BLOOD W I T H AGE IN NORMAL MICE
TABLE I11
37 -*- 1.0
(9)
(7)
36 :t: 0.7
8100 ± 600 (8)
6 weeks 12 weeks
7800 ± 700 (10)
2 years +
I year +
96 o r to h e m o p o i e t i c s u p p o r t i v e m i c r o e n v i r o n m e n t s [5]. M a t t h e w a n d C r o u s e r e p o r t e d t h a t s t r o m a l cells f r o m o l d m o u s e b o n e m a r r o w s u p p o r t e d C F U - S a n d G M - C F C in vitro better t h a n t h o s e f r o m y o u n g m o u s e m a r r o w [14]. O u r finding in BDF~ mice a p p e a r s to be in g o o d a g r e e m e n t with their findings. In c o n t r a s t , I n o u e et al. s t u d i e d the l o n g - t e r m survival o f stem cells t r a n s p l a n t e d into i r r a d i a t e d o l d mice a n d f o u n d a significant decrease in the self-renewal o f the stem cells in the senescent mice [15]. S h o r t - t e r m e v a l u a t i o n o f the stem cell c a p a c i t y for self-renewal, thus, a p p e a r s to be insufficient for the s t u d y o f the aging. R e m a r k a b l e f e a t u r e in this r e g a r d is a decrease in the n u m b e r o f splenic G M - C F C in BDF~ mice at the age o f 2 years. Such a decrease was even m o r e p r o f o u n d in S A M - P at the age o f 33 weeks. N u m b e r o f C F U - S d i d n o t s h o w a n y decrease o r r a t h e r increased with age in the spleen o f these mice. These findings m a y suggest a p o s s i b i l i t y t h a t the c a p a c i t y o f the d i f f e r e n t i a t i o n o f C F U - S to G M - C F C is r e d u c e d with age. A l t e r n a t i v e l y , it m a y suggest a r e d u c t i o n o f the h e m o p o i e t i c s u p p o r t i v e c a p a c i t y , at least o f d i f f e r e n t i a t i o n s u p p o r t i v e c a p a c ity, o f the splenic m i c r o e n v i r o n m e n t . Such a p r o f o u n d r e d u c t i o n o f the n u m b e r o f G M - C F C t o g e t h e r with a d e c r e a s e o f b o n e m a r r o w h e m o p o i e s i s m a y be r e s p o n s i b l e for the decrease in the n u m b e r o f p e r i p h e r a l leukocytes. S A M - P mice, which s h o w a g e - a s s o c i a t e d decrease in h e m o p o i e s i s , c o u l d p r o v i d e a g o o d e x p e r i m e n t a l m o d e l f o r t h e s t u d y o n the aging o f h e m o p o i e t i c system. ACKNOWLEDGMENT W e wish to t h a n k P r o f . T a k e d a o f Chest Disease Research Institute o f K y o t o U n i v e r s i t y for p r o v i d i n g S A M - P a n d -R mice a n d for helpful d i s c u s s i o n s . T h i s w o r k was s u p p o r t e d in p a r t b y a G r a n t - i n - A i d for Scientific Research f r o m the M i n i s t r y o f E d u c a t i o n , Science a n d C u l t u r e , J a p a n . REFERENCES 1 L.E. Botnik, E.C. Hannon, L. Obbagy and S. Hellman, The variation of hematopoietic stem cell self-renewal capacity as a function of age: further evidence for heterogeneity of the stem cell compartment. Blood, 60 (1982) 268 --271. 2 D.R. Boggs and K. Patrene, Hematopoiesis and aging V. A decline in hematocrit occurs in all aging female B6D2F~ mice. Exp. Aging Res., 12 (1986) 13 I-- 134. 3 C. Tojero, N.G. Testa and J.H. Hendry, Decline in cycling of granulocyte-macrophage colonyforming cells with increasing age in mice. Exp. Hematol., 17(1989) 66---67. 4 D.A. Ogden and H.S. Micklem, The fate of serially transplanted bone marrow cell populations from young and old donors. Transplantations, 22 (1976) 287--293. 5 T. Inoue and E.P. Cronkite, The influence of in vivo incubation of aged routine spleen colonyforming units on their proliferative capacity. Mech. Ageing Dev., 23 (1983) 177--190. 6 T. Takeda, M. Hoshokawa, S. Takeshita, M. lrino, K. Higuchi, T. Matsushita, Y. Tomita, K. Yasuhira, H. Hamamoto, K. Shimizu, K. lshii and T. Yamamuro, A new murine model of accelerated senescence. Mech. Ageing Dev., 17(1981) 183--194. 7 M. Hosokawa, R. Kasai, K. Higuchi, S. Takeshita, K. Shimizu, H. Hamamoto, A. Honma, M. Irino, K. Toda, A. Matsumura, M. Matsushita and K. Takeda, Grading score system: A method for evaluation of the degree of senescence in Senescence Accelerated Mouse (SAM). Mech. Ageing Dev., 26 (1984) 91--102.
97 A. Matsumura, K. Higuchi, K. Shimizu, M. Hosokawa, K. Hashimoto, K. Yasuhira and T. Takeda, A new amyloid fibril protein isolated from senescence-acceleratedmice. lab. Invest., 47 0982) 270---275. 9 S. Takeshita, M. Hosokawa, M. Irino, K. Higuchi, K. Shimizu, K. Yasuhira and T. Takeda, SponUmcous age-associated amyloidosis in senescence-acceleratedmouse (SAM). Mech. Ageing Dev., 20 (1982) 13--23. 10 K. Higuchi, A. Matsumura, A. Honma, S. Takeshita, K. Hashimoto, M. Husokawa, K. Yasuhira and T. Takeda, Systemic senile amyioid in Senescence-AcceleratedMouse: A unique fibril protein demonstrated in tissues from various organs by the unlabeled immunoperoxidase method. Lab. Invest., 48 (1983) 231--240. 11 M. Hosokawa, S. Takeshita, K. Higuchi, K. Shimizu, M. lrino, K. Toda, A. Honma, A. Matsumura, K. Yasuhira and T. Takeda, Cataract and ophthalmic lesion in senescence accelerated mouse (SAM). Morphology and incidence of senescence associated ophthalmic changes in mice. Exp. Eye Res., 38 (1984) 105--114. 12 T.R. Bradley and D. Metcalf, The growth of mouse bone marrow cells in vitro. Aust. J. Exp. Biol. Met/. Sci., 44 (1966)287--300. 13 J.E. Till and E.A. McCulloch, A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res., 14 (1961) 213--222. 14 K.I. Matthew and D.A. Crouse, An in vitro investigation of the hemopoietic microenvironment in young and aged mice. Mech. Ageing Dev., 17 (1981 ) 289--303. 15 T. Inoue, Y. Hirabayashi-Suzuki, H. Sasaki, K. Kuramoto, S. Matsuyama and M. Kanisawa, Hemopoietic stem cell growth and micro-environment of the senescent mice. Exp. HematoL, 17 0989) 602 (Abstr.). 8
89
ElsevierScientificPublishers Ireland Ltd.
AGE-RELATED DECREASE IN THE NUMBER OF HEMOPOIETIC STEM CELLS AND P R O G E N I T O R S IN SENESCENCE A C C E L E R A T E D MICE
HIROKO IZUMI-HISHA"*, YOSHITAKE ITO b, KENKICHI SUGIMOTO ~, H I D E H I K O OSHIMA" and K A Z U H I R O J. MORF "Department of Hygiene, Aichi Medical University and blnstitute for Aging Research, Aichi Medical University, Aichi and "Department of Biology, Faculty of Science, Niigata University, Niigata (Japan)
(ReceivedDecember14th, 1989) (Revision received June 8th, 1990)
SUMMARY Senescence accelerated mice (SAM-P) were used for the study o f the possible aging of hemopoiesis. The number of peripheral leukocytes decreased significantly with age, whereas hematocrit showed only a slight decrease. Although the number o f total nucleated cells in the bone marrow increased, the number of hemopoietic stem cells (CFU-S) as well as that of granulocyte-macrophage colony forming cells (GM-CFC) showed a decrease in old mice. A significant decrease in the number of GM-CFC was observed in the spleen o f old SAM-P mice, whereas no decrease was found in the number of CFU-S. Such a profound reduction o f the recruitment of GM-CFC from CFU-S in the spleen together with a reduction of bone marrow hemopoiesis may be responsible for the decrease in the number of peripheral leukocytes in the old mice. SAM-P mice could provide a good model for the study of the aging of hemopoietic system. K e y words: Aging; Hemopoiesis; Senescence accelerated mice; Hemopoietic stem
cells; Granulocyte macrophage progenitor cells INTRODUCTION Pluripotent hemopoietic stem cells (CFU-S) have a capacity of extensive selfrenewal. Aging of hemopoiesis, especially o f self-renewal capacity of CFU-S has been a matter o f debate for a long time. *Present address and to whom all correspondence should be addressed: First Department of Pathology, Kansai Medical University,Fumizono-cho1, Moriguchi-shi,Osaka, 570, Japan.
0047-6374/90/$03.50 Printed and Published in Ireland
© 1990ElsevierScientificPublishers Ireland Ltd.
90
A number of investigators reported that the self-renewal capacity as well as the number of CFU-S remained unchanged during the life span [1] although the hematocrit [2] and the population size of granulocyte-macrophage progenitor cells (GMCFC) in cycle [3] decreased with age. The repeated transplantation of hemopoietic stem cells into lethally-irradiated recipient mice resulted eventually in a loss of the ability to reconstitute hemopoietic system in host mice within 6 - - 8 serial transplantation [4]. lnoue and Cronkite [5] reported that the hemopoietic micro-environment of old mice had a reduced capacity to support the spleen colony formation derived from CFU-S with regard to the size of the spleen colonies. These contradictory findings might be due to the difference in the experimental procedures. Takeda et al. [6,71 established S A M - P / I - P / 5 mice (Senescence prone series of Senescence Accelerated Mouse), which have a short life span of about 9 months, from A K R / J mice. These mice were found to have an accelerated deposition of amyloid in their lung, spleen, liver, kidney etc., except brain and bone, after the age of 24 weeks [8--10]. Other characteristics of aging, such as cataract, periophthalmic lesion [11], increased skin coarseness, alopecia, increased lordokyphosis of spine and so on, were also observed. Such a manifestation of aging appears after normal growth, that is after 20--25 weeks and their aging advances in a accelerated manner. SAM-P mice would, therefore, be a suitable model of the study of the possible aging of the hemopoietic system. As controls, senescence resistance series (SAM R / I-R/3) were also established from A K R / J mice. Their life span is 13.3 months and their aging pattern is similar to other normal strains, when compared with that of SAM-P. The present study describes hemopoietic features of SAM-P mice in order to obtain fundamental data of age-related hemopoietic changes. MATERIALS AND M E T H O D S
Animals
A K R / J strain mice were originally donated from The Jackson Laboratory (Bar H a r b o r , ME). Senescence-prone series (SAM-P) were obtained by serial mating of the litters which had a short life span, by Takeda et al. [6,7]. Senescence-resistant series (SAM-R) were also established in 1981. These mice were reared under near specific pathogen-free (SPF) conditions and fed with a commercial diet (CE-2, Nihon Clea) and tap water ad libitum. The mice were used at the age of 8 weeks or 33 weeks, because the mice of these series have been well examined. Male SPF mice of BDF~ strain were purchased from the Charles River Breeding Laboratories (Japan) at the age of 8 weeks and maintained under SPF conditions. The mean life span of this strain is approximately 30 months at our animal facilities. Male SPF mice of ddY strain were obtained from the Shizuoka Laboratory Animal Center (Japan) and used at the age of 8 weeks or 33 weeks.
91
Peripheral blood Blood was obtained by cardiac or retro-orbital sinus puncture under ether anaesthesia and the number o f white blood cells (WBC) was counted for each mouse individually. Hematocrit (Hct) was also measured for each mouse.
Cell preparations Bone marrow and spleen cells were collected and pooled from 8 to I0 mice per each point. Single cell suspension were prepared and used for both CFU-S and GMCFC assays. In each experiment, the cells from young and old mice of the same strain were assayed simultaneously.
GM-CFC assay Number of GM-CFC in the bone marrow and the spleen was measured by the soft-agar culture technique using a modified method of Bradley and Metcalf [12]. Briefly, 1--2 x l05 bone marrow cells or 2 - - 4 × 106 spleen cells were plated into petri dishes containing 2 ml of Fischer's medium supplemented with 0.3070 agar, 25°70 horse serum and 10070 mouse lung-heart conditioned medium as colony-stimulating factor (CSF). A minimum of three replicate dishes were cultured for each point. The dishes were incubated for 7 days and the number of cell clusters consisted o f more than 50 cells were counted as colonies.
CFU-S assay Aliquots of the bone marrow or spleen cell suspension were used for the assay of CFU-S according to the method of Till and McCulloch [13]. For SAM-P mice, the appropriate number of bone marrow or spleen ceils from 6 to 10 mice were injected intravenously into 8-week-old SAM-R mice (10--13 per point) that had been irradiated with 8.0 Gy X-rays. The cells from other strains were injected i.v. into lethally irradiated (8.0 Gy for SAM-R, 9.5 Gy for BDF I and 8.0 Gy for ddY) mice. The recipient mice were sacrificed 10 days after the cell-injection and their spleens were fixed in Bouin's solution. The nodules on the surface of the spleen were counted after 1 day of fixation. RESULTS
Hemopoietic features o f SA M-P and SA M-R mice (a) Peripheral blood. The change in the values of WBC and Hct with age was investigated in both SAM-P and -R mice. Results are summarized in Table I. There was no difference either in WBC count or in Hct at the age of 8 and 16 weeks. The number of WBC at 33 weeks, however, reduced to half of that at 8 weeks in SAM-P mice (significant, P < 0.01). A slight decrease in the number of WBC was observed in SAM-R. The value of Hct decreased slightly at the age of 33 weeks only in SAM-P mice.
92
TABLE I A G E - A S S O C I A T E D C H A N G E S IN T H E F E A T U R E S O F P E R I P H E R A L
B L O O D IN S A M M I C E
8 Weeks
16 Weeks
33 Weeks
Whiteblood cells(/lal)
SAM-P SAM-R
6900 __. 1000 7200 :i: 800
7700 :t: 1000 7200 __. 1300
3500 :t: 200* 5700 _+ 700
Hematocrit (%)
SAM-P SAM-R
44 -*- 0.8 44 --- 0.8
43 :t: 1.0 43 --. 0.7
41.5 _ 1.5 45 --. 2.0
The values represent the m e a n ± S.E. o f 8 - - 1 0 mice. *Significant c h a n g e f r o m 8 weeks ( P < 0.01).
(b) Bone marrow andspleen. The change in the number of hemopoietic cells with age was also examined for bone marrow and spleen. The number of CFU-S and GMCFC in old SAM-P mice (33 week-old) expressed as the number per 105 cells were about 40070 of the number at 8 week-old mouse bone marrow (significant P < 0.01). As the number of total nucleated cells per femur increased with age, the net decrease of the stem cell population was about 30070 . A similar decrease in the number of GM-CFC was observed in SAM-R mice at 33 weeks (P < 0.01). No decrease was observed, however, in the number of CFU-S in these mice (Table II (a)). A marked decrease in the number of GM-CFC was observed in the spleen of both SAM-P and-R mice at 33 weeks (P < 0.01). The number of CFU-S, however, did not show any significant decrease in either of these mice (Table II (b)). The fact that a significant decrease with age of the number of GM-CFC occurs in SAM-R mice, makes it unsuitable to use SAM-R mice as 'normal' controls of SAMP mice. Effect of age on hemopoiesis o f normal mice (a) Peripheral blood. BDF~ and ddY mice were used as normal control and the changes in WBC count and Hct value were determined at various ages (Table III). No significant changes in the number of WBC or Hct values were observed in ddY mice at the age of 33 weeks. In BDF~ mice, Hct showed only slight decrease (insignificant). The number of WBC showed a slight increase at the age of 2 years compared to the number at the age of 8 weeks (insignificant). (b) Bone marrow andspleen. Numbers of GM-CFC and CFU-S of old mice were compared with those of 8 or 9 weeks. The ratio of 33 weeks:8 or 9 weeks is shown in Fig. 1. In order to facilitate the comparison with SAM-P mice, their results are also added to the figure. In ddY mice, numbers of GM-CFC and CFU-S per 105 bone marrow ceils at 33 weeks were 70--80°70 of the numbers of young mice. Similar decrease was also observed in BDF t mice at the age of I year + 10 weeks. The degree of the reduction, however, was smaller than that in SAM-P mice (insignificant). Since the number of
93
TABLE II A G E - A S S O C I A T E D C H A N G E S IN T H E NUMBER OF H E M O P O I E T I C C E L L P O P U L A T I O N IN BONE M A R R O W A N D S P L E E N OF SAM M I C E
(a) Bone marrow (1) SAM-P mice Nucleated cells CFU-S GM-CFC
/10~
/femur /10~
/femur (2) SAM-R mice Nucleated cells
CFU-S GM-CPC
/10~
/femur /los
/femur (b) Spleen (1) SAM-P mice Nueleatedcells CFU-S GM-CFC
/los
/spleen /los
/spleen (2) SAM-R mice
Nucleated cells /los CFU-S
/spleen
GM-CFC
/106
/spleen
8 Weeks
33 Weeks
Ratio o f 33 wk/8 wk
8.6 X los 25 ± 4 2150 ± 310 266 ± 25 22 876 ± 2167
17.8 x 9 1600 103 18 300
los ± 4" ± 730 ± 19" ± 3376
2.1 0.4 0.7 0.4 0.8
8.9 x los 29 ± 6 2554 ± 561 166 ± 5 14774 ± 445
14.4 x 27 3900 85 12200
106 ± 6 ± 821 ± 8* ± 1080
i.6 0.9 1.5 0.5 0.8
2.1 x los 58 ± 6 12243 ± 1323 200 ± 20 42 000 ± 4288
2.8 × 57 15 390 53 14 800
l 0s ± 5 ± 1700 ± 9* ± 2380
1.3 1.0 1.3 0.3 0.4
2.2 × los 21 ± 4 4620 ± 836 83 ± 15 18 260 ± 3380
3.1 × 20 8100 26 5700
los ± 6 ± 1562 ± 3* ± 992
1.4 1.4 1.8 0.3 0.3
The values represent the mean ± S.E. of 8 - - 10 mice. *Significant change from 8 weeks ( P < 0.01).
total bone marrow cells increased significantly with age, the total numbers o f GMCFC and CFU-S in bone marrow showed an increase at the age o f 1 year and 1 year + 10 weeks o f BDF. mice. This contrasts the marked decrease o f the stem cell populations at the age o f 33 weeks in SAM-P mice. N o significant decrease was observed in the number o f GM-CFC or CFU-S per 105 cells in the spleen o f ddY mice at the age o f 33 weeks (insignificant). Old mice of BDF~ strain (2 years old) showed a slight decrease in the number o f GM-CFC in the spleen, but it was not significant. DISCUSSION
In the first part o f this study, we examined the age-related change in hemopoiesis
94
!!-
(1) Bone ~
0
C4dle 8AM-P/1
ddY
(33vd~
BOF!
(33v4~)
(11mr)
( 11mar+lO vb.)
(2 yoars)
-
# t o t l d CleU.8 GM-(:PC
tOtM (]qJ-6 GM4:FC
total CF'J-8 OM.O:C: t,vld CFU-S GM-CFC
totad O:U-S
(2] ~:ie4m Ceils SM4-P/1
]
~
I
8DF~
r
,--,
8
0
~ tOial (]:U.4 G M . O ~ SC
total (]=U-S GM.O:C SC
: TOlal nuckmled c44t$ io~r fen~r ow Wieen
total CFQ-S GM.CFC SC
Fig. 1. Change in the number of hemopoietic cell population in bone marrow and spleen. Bone marrow and spleen cells were collected from mice of various ages and assayed for the number of GM-CFC and CFU-S. The results are expressed as percentage of the value at 8 weeks.
o f S A M - P and -R mice. No significant difference in the aging o f hemopoiesis was observed between these two groups o f mice. Deposition o f amyloid and other characteristics o f aging are also observed in S A M - R mice and therefore it seems that S A M - R mice finally shows aging o f the same substance as S A M - P mice. However, the time o f appearance o f aging and its degree in S A M - R mice are different f r o m those in S A M - P mice. It is shown that genetic profiles o f both mice are already different (unpublished data). It seems that their separation is not perfect yet as to hemopoiesis. Accordingly, we used old mice o f normal strains (ddY and BDF~) as a control o f S A M - P mice, because it is well-known that the aging pattern and the hemopoitic characteristics are n o r m a l in both strains. A significant decrease in the n u m b e r o f W B C was observed at 33 weeks o f S A M - P mice, whereas such an obvious decrease did not occur in these n o r m a l mice (Tables I, III). N u m b e r s o f C F U - S and G M - C F C per l0 s b o n e m a r r o w cells at the age o f 33 weeks o f S A M - P mice reduced to 40°70 o f those at 8 weeks (Fig. l-(l)). The total n u m b e r o f these cells also reduced as a result, although the total cellularity o f the bone m a r r o w increased with age. In n o r m a l mice, on the other hand, the decrease o f the stem cells and progenitor cells was small and even an increase o f the n u m b e r o f CFU-S and G M - C F C was observed at 1 year in BDF~ mice. Aging o f hemopoietic system is, if any, ascribed either to stem cells themselves [4]
43 ± 3.0
*The value represents the mean ± S.E. **Number of mice used.
(6)
Hematocrit
(~)
6700 -4- 600* (5)**
Whiteblood cells (/pl)
(7)
43 + 0.9
6300 ± 700 (7)
(8)
39 + 1.2
5800 ± 500 (9)
9 Weeks
8 Weeks
33 Weeks
BDFI
ddY
CHANGE IN THE FEATURES OF P E R I P H E R A L BLOOD W I T H AGE IN NORMAL MICE
TABLE I11
37 -*- 1.0
(9)
(7)
36 :t: 0.7
8100 ± 600 (8)
6 weeks 12 weeks
7800 ± 700 (10)
2 years +
I year +
96 o r to h e m o p o i e t i c s u p p o r t i v e m i c r o e n v i r o n m e n t s [5]. M a t t h e w a n d C r o u s e r e p o r t e d t h a t s t r o m a l cells f r o m o l d m o u s e b o n e m a r r o w s u p p o r t e d C F U - S a n d G M - C F C in vitro better t h a n t h o s e f r o m y o u n g m o u s e m a r r o w [14]. O u r finding in BDF~ mice a p p e a r s to be in g o o d a g r e e m e n t with their findings. In c o n t r a s t , I n o u e et al. s t u d i e d the l o n g - t e r m survival o f stem cells t r a n s p l a n t e d into i r r a d i a t e d o l d mice a n d f o u n d a significant decrease in the self-renewal o f the stem cells in the senescent mice [15]. S h o r t - t e r m e v a l u a t i o n o f the stem cell c a p a c i t y for self-renewal, thus, a p p e a r s to be insufficient for the s t u d y o f the aging. R e m a r k a b l e f e a t u r e in this r e g a r d is a decrease in the n u m b e r o f splenic G M - C F C in BDF~ mice at the age o f 2 years. Such a decrease was even m o r e p r o f o u n d in S A M - P at the age o f 33 weeks. N u m b e r o f C F U - S d i d n o t s h o w a n y decrease o r r a t h e r increased with age in the spleen o f these mice. These findings m a y suggest a p o s s i b i l i t y t h a t the c a p a c i t y o f the d i f f e r e n t i a t i o n o f C F U - S to G M - C F C is r e d u c e d with age. A l t e r n a t i v e l y , it m a y suggest a r e d u c t i o n o f the h e m o p o i e t i c s u p p o r t i v e c a p a c i t y , at least o f d i f f e r e n t i a t i o n s u p p o r t i v e c a p a c ity, o f the splenic m i c r o e n v i r o n m e n t . Such a p r o f o u n d r e d u c t i o n o f the n u m b e r o f G M - C F C t o g e t h e r with a d e c r e a s e o f b o n e m a r r o w h e m o p o i e s i s m a y be r e s p o n s i b l e for the decrease in the n u m b e r o f p e r i p h e r a l leukocytes. S A M - P mice, which s h o w a g e - a s s o c i a t e d decrease in h e m o p o i e s i s , c o u l d p r o v i d e a g o o d e x p e r i m e n t a l m o d e l f o r t h e s t u d y o n the aging o f h e m o p o i e t i c system. ACKNOWLEDGMENT W e wish to t h a n k P r o f . T a k e d a o f Chest Disease Research Institute o f K y o t o U n i v e r s i t y for p r o v i d i n g S A M - P a n d -R mice a n d for helpful d i s c u s s i o n s . T h i s w o r k was s u p p o r t e d in p a r t b y a G r a n t - i n - A i d for Scientific Research f r o m the M i n i s t r y o f E d u c a t i o n , Science a n d C u l t u r e , J a p a n . REFERENCES 1 L.E. Botnik, E.C. Hannon, L. Obbagy and S. Hellman, The variation of hematopoietic stem cell self-renewal capacity as a function of age: further evidence for heterogeneity of the stem cell compartment. Blood, 60 (1982) 268 --271. 2 D.R. Boggs and K. Patrene, Hematopoiesis and aging V. A decline in hematocrit occurs in all aging female B6D2F~ mice. Exp. Aging Res., 12 (1986) 13 I-- 134. 3 C. Tojero, N.G. Testa and J.H. Hendry, Decline in cycling of granulocyte-macrophage colonyforming cells with increasing age in mice. Exp. Hematol., 17(1989) 66---67. 4 D.A. Ogden and H.S. Micklem, The fate of serially transplanted bone marrow cell populations from young and old donors. Transplantations, 22 (1976) 287--293. 5 T. Inoue and E.P. Cronkite, The influence of in vivo incubation of aged routine spleen colonyforming units on their proliferative capacity. Mech. Ageing Dev., 23 (1983) 177--190. 6 T. Takeda, M. Hoshokawa, S. Takeshita, M. lrino, K. Higuchi, T. Matsushita, Y. Tomita, K. Yasuhira, H. Hamamoto, K. Shimizu, K. lshii and T. Yamamuro, A new murine model of accelerated senescence. Mech. Ageing Dev., 17(1981) 183--194. 7 M. Hosokawa, R. Kasai, K. Higuchi, S. Takeshita, K. Shimizu, H. Hamamoto, A. Honma, M. Irino, K. Toda, A. Matsumura, M. Matsushita and K. Takeda, Grading score system: A method for evaluation of the degree of senescence in Senescence Accelerated Mouse (SAM). Mech. Ageing Dev., 26 (1984) 91--102.
97 A. Matsumura, K. Higuchi, K. Shimizu, M. Hosokawa, K. Hashimoto, K. Yasuhira and T. Takeda, A new amyloid fibril protein isolated from senescence-acceleratedmice. lab. Invest., 47 0982) 270---275. 9 S. Takeshita, M. Hosokawa, M. Irino, K. Higuchi, K. Shimizu, K. Yasuhira and T. Takeda, SponUmcous age-associated amyloidosis in senescence-acceleratedmouse (SAM). Mech. Ageing Dev., 20 (1982) 13--23. 10 K. Higuchi, A. Matsumura, A. Honma, S. Takeshita, K. Hashimoto, M. Husokawa, K. Yasuhira and T. Takeda, Systemic senile amyioid in Senescence-AcceleratedMouse: A unique fibril protein demonstrated in tissues from various organs by the unlabeled immunoperoxidase method. Lab. Invest., 48 (1983) 231--240. 11 M. Hosokawa, S. Takeshita, K. Higuchi, K. Shimizu, M. lrino, K. Toda, A. Honma, A. Matsumura, K. Yasuhira and T. Takeda, Cataract and ophthalmic lesion in senescence accelerated mouse (SAM). Morphology and incidence of senescence associated ophthalmic changes in mice. Exp. Eye Res., 38 (1984) 105--114. 12 T.R. Bradley and D. Metcalf, The growth of mouse bone marrow cells in vitro. Aust. J. Exp. Biol. Met/. Sci., 44 (1966)287--300. 13 J.E. Till and E.A. McCulloch, A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res., 14 (1961) 213--222. 14 K.I. Matthew and D.A. Crouse, An in vitro investigation of the hemopoietic microenvironment in young and aged mice. Mech. Ageing Dev., 17 (1981 ) 289--303. 15 T. Inoue, Y. Hirabayashi-Suzuki, H. Sasaki, K. Kuramoto, S. Matsuyama and M. Kanisawa, Hemopoietic stem cell growth and micro-environment of the senescent mice. Exp. HematoL, 17 0989) 602 (Abstr.). 8
