Impairment of B lymphopoiesis in precocious aging (klotho) mice
Seiji Okada,
Toru Yoshida1,
Zhang Hong,
Genichiro Ishii2,
Masahiko Hatano,
Makoto Kuro-o3,
Yoko Nabeshima4,5,
Yo-ichi Nabeshima1,5 and
Takeshi Tokuhisa
Department of Developmental Genetics, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan
1 Department of Pathology and Tumor Biology, Graduate School of Medicine, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan
2 First Department of Pathology, Chiba University School of Medicine, Chiba 260-8670, Japan
3 Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9072, USA
4 Division of Molecular Genetics, National Institute of Neuroscience, NCNP, Kodaira 187-8502, Japan
5 Core Research for Evolutional Science and Technology (CREST), JRDC, Tokyo, Japan
Correspondence to:
T. Tokuhisa
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Abstract
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Inactivation of the klotho gene in mice results in multiple disorders that resemble human aging after 3 weeks of age. Because hematopoiesis, especially B lymphopoiesis, is affected in humans and mice by aging, we analyzed the hematopoietic state in homozygous klotho (kl/kl) mice. Thekl/kl mice showed thymic atrophy and a reduced number of splenocytes. These mice had almost the normal number of myeloid cells, erythroid cells, IL-3-responsive myeloid precursors and colony forming units in spleen (CFU-S) in bone marrow (BM), but had a substantially decreased number of B cells in BM and peripheral blood as compared with wild-type mice. IL-7-responsive B cell precursors and all of the maturation stages of B cells in BM were also reduced. However, the function of hematopoietic stem cells including their capacity of B lymphopoiesis in vivo and in vitro was normal. Early B cell development was also normal in neonates and young kl/kl mice until 2 weeks old without aging phenotypes. RT-PCR analysis revealed that the level of IL-7 gene expression was significantly reduced in freshly isolated kl/kl BM cells. However, injection of IL-7 in kl/kl mice could not rescue the B lymphopenia. These findings indicate that Klotho protein may regulate B lymphopoiesis via its influence on the hematopoietic microenvironment.
Keywords: hematopoiesis, IL-7, microenvironment
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Introduction
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Aging induces the deterioration of physiological functions necessary for the survival and fertility of an organism, and results in common age-related diseases such as arteriosclerosis, cancer, dementia and osteoporosis (1,2). Aging also affects the immune system (3,4). Both B cell function and development are modulated in humans and mice (3,5,6), and the generation of pre-B cells is impaired in bone marrow (BM) by aging (710). Since early B cell development is highly dependent on functions of stromal cells (11,12), it has been postulated that the impairment of pre-B cell development by aging is due to functional abnormalities of stromal cells including the production of IL-7 (5,9,13). However, Kirman et al. have recently shown that IL-7 production by BM cells from aged mice is not impaired and that apoptotic cell death of pre-B cells with a lower level of bcl-xL mRNA increases in BM from aged mice (14). Because aging is a complex process influenced by many genes (2), mechanisms of the age-associated alteration of early B cell development are not clearly understood yet.
A novel mouse mutant, termed klotho, that exhibits multiple disorders resembling human aging has recently been established (15). Defect of klotho gene expression (kl/kl) in mice results in multiple age-related disorders such as arteriosclerosis, osteoporosis, pulmonary emphysema, short lifespan and infertility after 3 weeks of age. Furthermore, kl/kl mice showed atrophy of the anterior pituitary gland which regulates the other endocrine organs. These results suggest that the klotho gene product is involved in the suppression of multiple aging phenotypes at a young age. The klotho gene encodes a novel cell surface protein of 1014 amino acids that has a cleavage site to generate a secreted form (1517). The extracellular domain consists of two internal repeats which exhibit 2040% sequence identity to ß-glucosidases of bacteria and plants as well as to mammalian lactase glycosylceramidase. The klotho gene is expressed in various organs, but not in lymphatic organs including BM, thymus and spleen. Because some organs without endogenous expression of the klotho gene were severely affected in kl/kl mice, it is considered that the secreted form of Klotho protein mainly works as a humoral factor to protect against precocious aging (15,17). Since kl/kl mice show the multiple phenotypes resembling human aging caused by a single gene mutation, they are a powerful tool to understand the mechanisms of age-related disorders.
In the present study, we analyzed the hematopoietic status of kl/kl mice with precocious aging. Early B cell development was impaired in BM from kl/kl mice, although the function of hematopoietic stem cells, the numbers of myeloid progenitors and myeloid cells was normal in BM from kl/kl mice. IL-7 production in vivo by BM from kl/kl mice was very low. However, administration of IL-7 in kl/kl mice could not rescue the B lymphopenia. We discuss abnormalities of the hematopoietic microenvironment in BM from kl/kl mice.
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Methods
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Mice
Heterozygous klotho mutant (kl/+) mice (C3H) were maintained in our animal facility and intercrossed, and the resulting littermates were used for this study. Genotypes were confirmed by Southern blot analysis of tail DNA (15). SCID mice were purchased from Japan SLC (Shizuoka, Japan).
Hematopoietic growth factors
Murine rIL-3 and rIL-7 were provided by Dr Tetsuo Sudo (Toray Industries, Kamakura, Japan). The concentration of the cytokines used in vitro was as follows: rIL-3, 200 U/ml; rIL-7, 20 U/ml. As for the in vivo injection study, murine rIL-7 purchased from Genzyme (Cambridge, MA) was used at 500 ng/day.
mAb and FACS analysis
Biotinylated, FITC-conjugated or phycoerythrin (PE)-conjugated mAb against B220 (RA3-6B2), CD43 (S7), CD24 (HSA; J11d), BP-1 (Ly-51; 6C3), IgM, Mac-1 (M1/70), Gr-1 (RB6-8C5), Thy1.2 (53-2.1), CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.72), CD5 (Ly-1; 53-7.3) or TER119 (Ly-76) were purchased from PharMingen (San Diego, CA). PE-conjugated anti-Sca-1 (Ly 6A/E) mAb was purchased from PharMingen, and FITC-conjugated anti-c-kit (CD117) mAb was a gift from Dr S.-I. Nishikawa (Kyoto University, Japan) and used to purify the primitive hematopoietic stem cell fraction (18). Biotinylated mAb were visualized using streptavidinallophycocyanin or streptavidinCyChrome (PharMingen).
Cell suspensions were treated with ACK lysing buffer (0.155 M ammonium chloride, 0.1 M disodium EDTA and 0.01 M potassium bicarbonate) to lyse erythrocytes before staining. Single-cell suspensions were prepared in staining medium (PBS with 3% FCS and 0.1% sodium azide) and were stained with the mAb described above. After 20 min incubation on ice, cells were washed twice with staining medium and resuspended in staining medium supplemented with propidium iodide (PI; 1 µg/ml). Stained cells were analyzed by a FACSCalibur (Becton Dickinson, San Jose, CA).
B-lineage cells were resolved into various fractions (AF) based on the technique initially reported by Hardy et al. (19,20). Fraction A (B220+CD43+CD24) cells were distinguished from Fraction B + C (B220+CD43+CD24+) cells on the basis of CD24 expression. Fraction D cells were defined as B220+CD43IgM cells and Fraction E + F were defined as B220+IgM+ cells.
Isolation of Linc-kit+Sca-1+ BM cells and co-culture with stromal cells
Total BM cells from kl/kl mice or littermates were stained with a cocktail of biotinylated mAb against lineage markers (Lin; B220, Mac-1, Gr-1, CD4, CD8 and TER119) for 20 min at 4°C. After washing the cells 3 times with staining medium, the cells were treated with streptavidin-conjugated immunomagnetic beads (BioMag; Perceptive Diagnostics, Cambridge, MA) for 30 min to remove Lin highly positive cells. The remaining cells were collected and stained with PEanti-Sca-1 mAb, FITCanti-c-kit mAb and streptavidinCyChrome for 20 min at 4°C. After washing, the cells were resuspended in staining medium supplemented with PI (1 µg/ml). Stained cells were analyzed by FACS Vantage (Becton Dickinson), and the Linc-kit+Sca-1+ cells were sorted and used as a primitive hematopoietic stem cell fraction (18). Murine stromal cell line, OP-9 (21) was obtained from RIKEN cell bank (Tsukuba, Japan). OP-9 was seeded in six-well plates (Becton Dickinson Labware, Lincoln Park, NJ) 1 day before co-culture. Whole BM cells or sorted Linc-kit+Sca-1+ BM cells were cultured on a OP-9 stromal layer with 3 ml of RPMI 1640 containing rIL-7 (20 U/ml), 10% heat-inactivated FCS and 5x105 M 2-mercaptoethanol.
In vitro colony assay
Methylcellulose culture was carried out using a modified method of Iscove et al. (2224). Briefly, 1 ml of culture medium contained an adequate number of total BM cells, 1.2% methylcellulose (Shin-etsu Chemical, Tokyo, Japan),
-medium (Gibco, Grand Island, NY), 30% FCS (Sigma, St Louis, MO), 1% deionized BSA (Sigma), 0.1 mM 2-mercaptoethanol (Eastman Organic Chemical, Rochester, NY) and the appropriate concentration of growth factors. The cultures were prepared in 35 mm non-tissue culture dishes (Becton Dickinson Labware) and incubated at 37°C in a humidified atmosphere of 5% CO2. The number of colonies was scored after 7 days of culture using an inverted microscope.
Colony forming unit in spleen (CFU-S) assay
Female wild-type mice were X-irradiated (9.5 Gy). After 24 h, the mice were injected i.v. with 1x105 cells suspended in PBS. Mice were anesthetized and sacrificed by cervical dislocation at 12 days after injection. The spleens were removed, fixed in Bouin's solution and colonies were counted (25).
Long-term reconstitution assay
Female wild-type mice and SCID mice were X-irradiated (9.5 and 2.0 Gy respectively). After 24 h, male BM cells (1x106) suspended in PBS were injected into the tail vein of female recipients. Peripheral blood (PB) of SCID mice was obtained from the retro-orbital venous plexus 20 weeks after transplantation and lysed with ACK lysing buffer. The samples were then used for flow cytometric analysis. The wild-type recipient mice were anesthetized and sacrificed by cervical dislocation at 20 weeks after injection. Thymus, spleen and BM were obtained, and genomic DNA was extracted from these organs. DNA samples (100 ng) were used for the PCR reaction. The sequence of the Y chromosome-specific primers (pY2) was 5'-GCATTTGCCTGTCAGAGAGAG-3' and 5'-ACTGCTGCTGCTTTCCAACTA-3' (26,27). After an initial 7 min incubation at 95°C, the 30 cycles of PCR reactions were carried out using the following conditions: denaturation at 95°C for 1.0 min, annealing at 62°C for 2.0 min and polymerization at 72°C for 3.0 min. The amplified DNA was analyzed by agarose gel electrophoresis and stained with ethidium bromide.
Histological analysis
Femurs were fixed in 10% neutral-buffered formalin and then treated for decalcification using formic acid. Paraffin-embedded tissues were subjected to hematoxylin & eosin staining.
RT-PCR analysis
Total RNA was extracted from BM cells using an ISOGEN total RNA isolating kit (Waco, Tokyo, Japan). RNAs were reverse-transcribed using Superscript (Life Technologies, Grand Island, NY) and oligo(dT) (Pharmacia, Piscataway, NJ), in a final volume of 20 µl, and 1 µl of cDNAs was used for PCR. After an initial 7 min incubation at 95°C, the 30 cycles of PCR reactions were carried out using the following conditions. IL-7, macrophage colony stimulating factor (M-CSF) and stem cell factor (SCF) cDNA: denaturation at 95°C for 1.5 min, annealing at 55°C for 1.5 min, and polymerization at 72°C for 1.5 min. G3PDH cDNA: denaturation at 95°C for 1 min, annealing at 60°C for 1 min and polymerization at 72°C for 1 min. PCR primers for the cDNA amplification were as follows: IL-7 primers, 5'-ACATCATCTGAGTGCCACA-3' and 5'-CTCTCAGTAGTCTCTTTAG-3' (28); M-CSF primers, 5'-CAGATCAAGGAAGACAACCG-3' and 5'-ATGGTACATCCACGCTGCGT-3' (29); SCF primers, 5'-GACTGTGTGCTCTCTTCAAC-3' and 5'-CTTGCAAAACCTCCAGAGTC-3' (29); and the G3PDH primers, 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3' and 5'-CATGTAGGCCATGAGGTCCACCAC-3' (30). The PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide.
IL-7 treatment in vivo
Female 4-week-old kl/kl and +/+ mice were given s.c. 500 ng/day of mouse rIL-7 dissolved in PBS containing 0.1% BSA for 6 days (31). Control mice were treated with vehicle solution (PBS/0.1% BSA) according to the same protocol. The mice were anesthetized and sacrificed by cervical dislocation on day 7. Flow cytometric analysis was performed with BM cells collected from the vehicle- and the IL-7-treated mice.
Statistical analysis
Data were analyzed using a single-tailed Student's t-test. Data are presented as mean ± SD.
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Results
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Hematopoiesis in homozygous kl/kl mice with precocious aging
Many aging phenotypes develop in homozygous kl/kl mice after 3 weeks of age (15). To examine hematopoiesis in kl/kl mice with aging phenotypes, percentages of T (Thy1+) cells, B (B220+) cells, myeloid (Mac-1+) cells and erythroid (TER119+) cells were analyzed in hematopoietic organs from kl/kl mice at 6 weeks by FACS (Fig. 1
). It is noteworthy that the B cell:T cell ratio in the kl/kl spleen was normal (Fig. 1B
), although the B cell:T cell ratio in the kl/kl PB was altered (Fig. 1A
). Since the thymus was atrophic and was almost not detected in the 6-week-old kl/kl mice as described (15), the cellularities were analyzed in PB, spleen and BM from kl/kl mice (Table 1
). The number of leukocytes in PB from kl/kl mice was ~50% of that from control littermates (+/+ mice) and this reduction was mainly due to the lower number of B cells (Fig. 1A
). The number of total nucleated cells in spleen from kl/kl mice was significantly lower than that from +/+ mice (P < 0.001). The number of total nucleated cells in BM from kl/kl mice was slightly lower than that from +/+ mice (P = 0.03). The number of B lymphocytes in the kl/kl BM was significantly lower than that in the +/+ BM (P < 0.001), whereas the number of myeloid cells, erythroid cells and T cells in the kl/kl BM was slightly higher without statistical significance. Interestingly, the percentage of TER119+ erythroid cells in the kl/kl spleen was drastically reduced (16.2 versus 7.4%), whereas it was normal or slightly elevated in the kl/kl BM. These results indicate that B cell development is impaired in BM from kl/kl mice with precocious aging. On the other hand, no significant difference in the proportion of CD5+ B cells (B-I cells) in the peritoneal cavity of kl/kl mice was observed (Fig. 1C
).

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Fig. 1. Flow cytometric analysis of lymphocytes from 6-week-old kl/kl mice. One representative FACS profile from six mice is presented for PB (A), spleen (B) and peritoneal cavity (C) of +/+ and kl/kl mice.
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Analysis of B progenitors in BM from kl/kl mice
Development of early B-lineage cells in BM from kl/kl mice at 6 weeks was further analyzed by FACS with cell surface staining for stage-specific cell surface markers. Pro-B, pre-B and mature B cells were thus defined as B220+CD43+IgM (Fraction AC), B220+CD43IgM (Fraction D) and B220+IgM+ (Fraction E + F) B cells respectively (19). The number of each stage of B-lineage cells in kl/kl mice was lower than that in +/+ mice (Fig. 2A
) and reduction of the pre-B cells was more than that of the mature (IgM+) B cells. Subpopulations among B220+CD43+IgM (Fraction AC) pro-B cells were further analyzed by their expression of BP-1 or CD24 to distinguish between Fraction A and Fraction B + C (Fig. 2B
). The percentage of BP-1+ or CD24high subpopulations among pro-B cells was apparently lower in kl/kl mice, although all stages of B subpopulations were reduced in kl/kl mice. These results were summarized in Table 2
.

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Fig. 2. Flow cytometric analysis of B-lineage cells in BM from 6-week-old kl/kl mice. (A) BM cells from +/+ and kl/kl mice were stained with mAb for anti-IgMFITC, anti-B220allophycocyanin and anti-CD43PE. (B) B220+CD43+ BM cells were further analyzed with expression of BP-1 and CD24 (HSA). Both BP-1+ and CD24+ fraction within B220+CD43+ BM cells were severely reduced. The number in each square represents the percentage within each subset. One representative result from six mice.
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The number of clonogenic progenitors in BM from kl/kl mice at 6 weeks was examined by the in vitro colony assay and the in vivo CFU-S assay (Table 3
). The number of IL-7-responsive clonogenic progenitors in kl/kl mice was reduced up to 5% of that in +/+ mice. These results are consistent with the reduction in the number of pro-B cells (Fraction B + C) in kl/kl mice, since this population contains the vast majority of IL-7-responsive B-lineage cells (32). In contrast, the number of IL-3-responsive clonogenic progenitors (committed myeloid progenitors) and day 12 CFU-S (more primitive hematopoietic stem cell fraction) in kl/kl mice was almost the same as that in +/+ mice, suggesting the presence of primitive hematopoietic stem cells in BM from kl/kl mice with aging phenotypes.
In order to examine the capacity for B lymphopoiesis by primitive hematopoietic stem cells, total BM cells or Linc-kit+Sca-1+ BM cells (primitive hematopoietic stem cell fraction) of kl/kl mice at 6 weeks were cultured in the presence of confluent layers of OP-9 plus rIL-7 (20 U/ml) (Fig. 3
). When total BM cells were used, the emergence of B cells (both B220+IgM immature B cells and B220+IgM+ mature B cells) was delayed in kl/kl mice compared with that in +/+ mice. When Linc-kit+Sca-1+ BM cells were used, the emergence of B cells in kl/kl and +/+ mice was almost the same, indicating a normal capacity of B lymphopoiesis by hematopoietic stem cells in BM from kl/kl mice.

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Fig. 3. In vitro analysis of B lymphopoiesis by BM cells from 6-week-old +/+ and kl/kl mice. Whole BM or Linc-kit+Sca-1+ BM cells were cultured over a confluent layer of OP-9 stromal cells supplemented with rIL-7 for 19 days. Expression of B220 and IgM on cultured BM cells was examined at different time intervals. Non-adherent cells harvested from four dishes by gentle pipetting at every medium change were rinsed once with medium, pooled and counted. The data presented are representative of two independent experiments.
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In vivo B lymphopoiesis by hematopoietic stem cells from kl/kl mice
In order to confirm the capacity of B lymphopoiesis in vivo by hematopoietic stem cells from kl/kl mice, we examined the long-term reconstitution of BM cells. We injected BM cells (2x106) obtained from 6-week-old male kl/kl mice into lethally irradiated female wild-type mice. All of the BM transplanted mice survived for 20 weeks, whereas all of the irradiated mice without BM transplantation were dead within 2 weeks. After 20 weeks, mice were sacrificed and DNA was extracted from BM, spleen and thymus. PCR study using a mouse Y chromosome-specific primer (pY2) (26,27) clearly showed that BM, spleen and thymus were reconstituted with cells derived from kl/kl mice (Fig. 4A
). Next, we injected kl/kl BM cells (2x106) into 2 Gy irradiated SCID mice which have no mature lymphocytes in PB. After 20 weeks, PB was obtained and stained with anti-CD3FITC and anti-B220PE respectively. As shown in Fig. 4
(B), the frequency of B220+ B lymphocytes [16.1 ± 7.2% (+/+) versus 15.6 ± 4.0% (kl/kl), n = 3, P > 0.05] and CD3+ T lymphocytes [40.4 ± 10.2% (+/+) versus 39.0 ± 12.7% (kl/kl), n = 3, P > 0.05] in kl/kl BM reconstituted SCID mice was comparable to the frequency of those populations in wild-type BM reconstituted SCID mice, whereas irradiated SCID mice without BM transplantation had few B and T lymphocytes in PB (33). Although these assays are not quantitative, these observations indicate that hematopoietic stem cells from kl/kl mice have radioprotective activity, have ability to reconstitute hematopoietic organs and are able to differentiate into B lymphocytes in appropriate circumstances.

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Fig. 4. In vivo analysis of B lymphopoiesis by BM cells from 6-week-old kl/kl mice. BM cells (2x106) from male +/+ or kl/kl mice were transplanted into the lethally irradiated (9.5 Gy) female +/+ mice (A) or irradiated (2 Gy) SCID mice (B). (A) Twenty weeks later, BM, spleen (Sp) and thymus (T) were harvested from each mouse and analyzed for the presence of donor-derived male cells by PCR. M, BM cells from +/+ male mice; F, BM cells from +/+ female mice. (B) Twenty weeks later, PB was obtained from each mouse, stained with mAb to CD3FITC and B220PE respectively, and analyzed for the presence of donor-derived lymphocytes. The number in each square represents the percentage within each subset. One representative result from three mice.
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Normal B lymphopoiesis was further confirmed in BM from young kl/kl mice. Since many aging phenotypes are displayed in kl/kl mice after 3 weeks (15), we examined B lymphopoiesis in liver from newborn kl/kl mice and in BM from kl/kl mice at 2 weeks. As shown in Fig. 5
, normal B cell development was observed in those organs from kl/kl mice, suggesting that the klotho gene product is not involved in B lymphopoiesis at least until 2 weeks after birth and that B lymphopenia in BM from kl/kl mice is due to the secondary effect of aging phenotypes.

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Fig. 5. Flow cytometric analysis of newborn liver and BM cells from kl/kl mice. The number in each square represents the percentage within the immature (B220+IgM) and mature (B220+IgM+) B cells. One representative result from five to eight mice.
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IL-7 production of stromal cells in BM from kl/kl mice
To examine abnormalities in BM from kl/kl mice, the structure of BM in femurs from kl/kl mice at 6 weeks was histologically analyzed (Fig. 6
). The thickness of cortical bone was thinner than that from +/+ mice as described (15). The BM space of femurs from kl/kl mice was retained or slightly extended. BM cellularity was not reduced (normocelular BM) and fatty changes which are often seen with aging were not observed in kl/kl mice. Furthermore, myeloid cells but not mononuclear cells were prominent in kl/kl mice, which is compatible with B lymphopenia in BM from kl/kl mice shown in Tables 1 and 2
.

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Fig. 6. Histological analysis of BM from 6-week-old +/+ and kl/kl mice. Hematoxylin & eosin-stained sections of femur are shown. (A and B) Original magnification x2. (C and D) Original magnification x100.
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As IL-7 and SCF derived from stromal cells are known to be essential for early B cell development in BM (11,12), we measured expression of IL-7, SCF and M-CSF mRNA in BM cells from kl/kl mice at 6 weeks by a semiquantitative RT-PCR method. As shown in Fig. 7
, expression of IL-7 mRNA in BM cells from kl/kl mice was significantly lower than that from +/+ mice. On the other hand, expression of SCF and M-CSF mRNA from the kl/kl BM was identical to that from the +/+ BM, which is consistent with the fact that myeloid lineage differentiation is not affected in kl/kl mice (Tables 1 and 3
).

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Fig. 7. Expression of IL-7 mRNA in BM of 6-week-old kl/kl mice. Expression of IL-7, SCF and M-CSF mRNAs was measured by semiquantitative RT-PCR in freshly isolated BM cells from +/+ and kl/kl. cDNAs serially diluted 8 times were used for PCR and starting amounts of cDNAs for the dilution were normalized by the amount of G3PDH. M, size marker. One representative result from three mice.
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We then tried to rescue B lymphopenia in BM from kl/kl mice at 4 weeks by administering IL-7 (Table 4
). When +/+ mice were treated with rIL-7 for 6 days, the proportion of Fraction A + B + C and Fraction D in BM was significantly increased compared with the vehicle-treated mice (P < 0.05 and P < 0.01 respectively). However, B-lineage cells were not developed in kl/kl mice by IL-7 treatment. Thus, IL-7 responsiveness of B-lineage cells is impaired in vivo in kl/kl mice.
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Discussion
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In this study, we examined the hematopoietic state of kl/kl mice with precocious aging in vivo and in vitro, focusing on the possible role of Klotho protein in proliferation and differentiation of hematopoietic stem cells. Early B cell development was preferentially impaired in BM from kl/kl mice, whereas myeloid cell development was not affected. However, hematopoietic stem cells in adult kl/kl mice were able to differentiate into mature B cells both in SCID mice in vivo, and in the presence of OP-9 stromal cells and IL-7 in vitro, and B cell development was not perturbed in neonates and young kl/kl mice without aging phenotypes. These results suggest that Klotho protein is not essential for B lymphopoiesis. This is supported by the evidence that B cell development is maintained by a nearly protein-free defined medium, i.e. Klotho protein-negative condition in vitro (34). Therefore, Klotho protein is considered to play a role in B cell differentiation indirectly and possibly to influence the hematopoietic microenvironment in BM.
The hematopoietic microenvironment in BM plays a critical role in B lymphopoiesis (11,12,35). It consists of a variety of cells including macrophages, fibroblasts, endothelial cells, pre-adipocytes, osteoblasts, osteoclasts and hematopoietic stem cells themselves. It is a complex, three-dimensional structure composed of these cells and abundant extracellular matrix (36), and these cells interact with each other to maintain constitutive hematopoiesis (36,37). In particular, the functional balance between osteoclasts and osteoblasts plays a critical role in bone remodeling and maintenance of hematopoietic microenvironment in BM including early B lymphopoiesis (38). Previously we showed that B lymphopoiesis is impaired in osteopetrotic c-fos-deficient mice and c-src-deficient mice (39), and concluded that the defect in B cell development in these mice is due to abnormalities of the microenvironment in BM and spleen as a consequence of osteopetrosis. These mice show the extramedullary hematopoiesis in spleen and selective impairment of B cell development, suggesting that the splenic hematopoietic microenvironment without osteoclasts and osteoblasts may not be suitable for B cell development. Since the hallmark of osteoporosis is a reduction in skeletal mass caused by an imbalance between bone resorption and bone formation (40), osteoporosis may also perturb the hematopoietic microenvironment in BM. As B lymphopoiesis is affected by aging (710), changes of the hematopoietic microenvironment caused by osteoporosis are considered to be critical for B lymphopoiesis.
As the development of early B cell precursors depends on IL-7 produced by stromal cells (11,12,35,4143), it was reasonable to ascribe the age-associated decline in the number of pre-B cells to a decreased IL-7 production (9), release (13) or responsiveness in BM (10). We found that expression of IL-7 mRNA by fresh BM cells was reduced in kl/kl mice, but that stromal cells generated in Whitlock Witte-type long-term BM cultures from kl/kl mice could maintain IL-7 mRNA expression and B lymphopoiesis (data not shown). Since expression of SCF and M-CSF mRNA and myeloid differentiation were not impaired in kl/kl mice, only the function of IL-7-producing subsets of stromal cells might be suppressed in vivo and IL-7 production of the subsets was recovered by in vitro culture. The reduced expression of BP-1 in the CD43+ compartment in kl/kl BM (Fig. 2B
) was consistent with a reduction in IL-7 availability in kl/kl BM, since IL-7 can up-regulate BP-1 expression on pro-B cells and induce proliferation of early B-lineage cells (44). Furthermore, IL-7 administration could not rescue the B lymphopenia in vivo (Table 4
), suggesting that IL-7 responsiveness is also impaired in BM from kl/kl mice.
Although IL-7 is required for development of pre-B cells (35,41), it is not sufficient for survival and proliferation of B-lineage cells (35,42,43,45). Stromal cell contact is essential for B lymphopoiesis (11,12,42,43). Stromal cells are thought to elaborate many important factors for B lymphopoiesis (35,45,46) and molecules that promote adhesion of B cell precursors to stromal cells (47). As B cell precursors from kl/kl mice can proliferate in response to IL-7 in vitro (Fig. 3
), it is possible that negative regulators are produced by stromal cells in vivo (48,49). In addition, the three-dimensional structure of BM plays a very important role in the in vivo organization of B lymphopoiesis. Progenitor cells are initially located in or near the endosteum of the BM and begin to differentiate in the peripheral region of BM (50). Thus, changes of these structures due to klotho deficiency (possible osteoporosis) may cause the altered B lymphopoiesis. Therefore, possible mechanisms to inhibit B lymphopoiesis in BM by klotho deficiency are as follows: (i) loss of Klotho protein modulates positive signals for B lymphopoiesis, (ii) Klotho protein may control synthesis of negative regulators and (iii) loss of Klotho protein changes the three-dimensional structure of hematopoietic microenvironment or suppresses some specific cell types important for B lymphopoiesis.
kl/kl mice show the abnormalities in the pituitary gland (15). The pituitary gland controls many other endocrine glands and is thus indirectly involved in many physiological functions (51) and its importance for the immune system has recently been recognized (52,53). Furthermore, it is known that only thyroid hormone is critical for normal B cell development among pituitary gland-related hormones (54,55). Although the free thyroxine level (0.68 ± 0.51 ng/dl) in serum of kl/kl mice was significantly lower than that (1.20 ± 0.19 ng/dl) of wild-type mice as expected (mean ± SD of four pools of four mice each, P < 0.01), thyroxine treatment failed to restore the frequency of B cell populations to normal (data not shown). These results indicate that hypothyroidism is not essential for B lymphopenia in kl/kl mice. One explanation for the observation is that Klotho protein may play an important role in the responsiveness to thyroxine and that the decreased responsiveness may be a part of the reason for B lymphopenia in kl/kl mice.
In contrast to the reduction of B lymphocyte progenitors, the frequency of myeloid progenitors and day 12 CFU-S did not decrease in BM from kl/kl mice. Furthermore, BM cells from kl/kl mice could reconstitute hematopoiesis in the lethally irradiated mice (Fig. 4A
), indicating that primitive hematopoietic stem cells with long-term repopulating ability and myeloid cell differentiation appear to be normal in kl/kl mice. The function of hematopoietic stem cells is known to change with age (56,57). The proliferative potential of BM cells from aged mice is greater than or equal to that of BM cells from young mice (5861). Recently, Morrison et al. showed that the frequency of hematopoietic stem cells and myeloid committed progenitors and the ratio of hematopoietic stem cells in cell cycling were increasing with age (62). However, it is unknown whether these changes are determined intrinsically or caused by changes of the hematopoietic microenvironment in aged mice. Since expression of the klotho gene cannot be detected in lymphocytes, BM cells and bone, the secreted isoform may play a major role in hematopoiesis (1517). Further investigation is necessary to determine a receptor for the secreted form of Klotho protein to understand not only the mechanisms of aging in kl/kl mice but also the function of hematopoietic microenvironment in B lymphopoiesis.
In conclusion, we showed that adult kl/kl mice suffer from severe B lymphopenia according to the impaired B cell development in BM. Since the functions of hematopoietic stem cells are normal, changes of the hematopoietic microenvironment in BM may be responsible for this phenomenon. Further studies including target cells for the secreted form of Klotho protein in BM clarify the mechanisms of B lymphopenia in kl/kl mice.
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Acknowledgments
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This work was partly supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan, the Mochida Memorial Foundation for Medical and Pharmaceutical Research and the Meiji Life Foundation of Health and Welfare. We thank Drs A. M. Stall and T. Suda for helpful discussions, Dr T. Sudo for reagents, Ms N. Fujita for secretarial services, and Ms H. Satake for skillful technical assistance.
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Abbreviations
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BM bone marrow |
CFU-S colony forming unit in spleen |
kl/kl mice homozygous klotho mice |
Lin lineage markers |
M-CSF macrophage colony stimulating factor |
PB peripheral blood |
PE phycoerythrin |
PI propidium iodide |
SCF stem cell factor |
+/+ mice control littermate mice. |
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Notes
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Transmitting editor: C. J. Paige
Received 20 August 1999,
accepted 18 February 2000.
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References
|
---|
-
Kirkwood, T. B. 1996. Human senescence. Bioessays 18:1009.[ISI][Medline]
-
Jazwinski, S. M. 1997. Longevity, genes, and aging. Science 273:54.[Abstract]
-
Miller, R. A. 1996. The aging immune system: primer and prospectus. Science 273:70.[Abstract]
-
Hodes, R. J. 1997. Aging and the immune system. Immunol. Rev. 160:5.[ISI][Medline]
-
Klinman, N. R. and Kline, G. H. 1997. The B-cell biology of aging. Immunol. Rev. 160:103.[ISI][Medline]
-
Sansoni, P., Cossarizza, A., Brianti, V., Fagnoni F., Snelli, G., Monti D., Marcato, A., Passeri, G., Ortolani, C., Forti, E., Fagiolo, U., Passeri, M. and Franceschi, C. 1993. Lymphocyte subsets and natural killer cell activity in healthy old people and centenarians. Blood 82:2767.[Abstract]
-
Rolink, A., Haasner, D., Nishikawa, S. and Melchers, F. 1993. Changes in frequencies of clonable pre B cells during life in different lymphoid organs of mice. Blood 81:2290.[Abstract]
-
Zharhary, D. 1988. Age-related changes in the capability of the bone marrow to generate B cells. J. Immunol. 1988 141:1863.
-
Stephan, R. P., Sanders, V. M. and Witte, P. L. 1996. Stage-specific alterations in murine B lymphopoiesis with age. Int. Immunol. 8:509.[Abstract]
-
Stephan, R. P., Lill-Elghanian, D. A. and Witte, P. L. 1997. Development of B cells in aged mice: decline in the ability of pro-B cells to respond to IL-7 but not to other growth factors. J. Immunol. 158:1598.[Abstract]
-
Kincade, P. W. 1991. Molecular interactions between stromal cells and B lymphocyte precursors. Semin. Immunol. 3:379.[Medline]
-
Nishikawa, S. I., Era, T., Ogawa, M., Nishikawa, S., Ohno, N., Hayashi, S. I. and Kunisada, T. 1992. Control of intramarrow B-cell genesis by stromal cell-derived molecules. Curr. Top. Microbiol. Immunol. 182:27.[Medline]
-
Stephan, R. P., Reilly, C. R. and Witte, P. L. 1998. Impaired ability of bone marrow stromal cells to support B-lymphopoiesis with age. Blood 91:75.[Abstract/Free Full Text]
-
Kirman, I., Zhao, K., Wang, Y., Szabo, P., Telford, W. and Weksler, M. E. 1998. Increased apoptosis of bone marrow pre-B cells in old mice associated with their low number. Int. Immunol. 10:1385.[Abstract]
-
Kuro-o, M., Matsumura, Y., Aizawa, H., Kawaguchi, H., Suga, T., Utsugi, T., Ohyama, Y., Kurabayashi, M., Kaname, T., Kume, E., Iwasaki, H., Iida, A., Shiraki-Iida, T., Nishikawa, S., Nagai, R. and Nabeshima, Y. 1997. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 390:45.[ISI][Medline]
-
Matsumura, Y., Aizawa, H., Shiraki-Iida, T., Nagai, R., Kuro-o, M. and Nabeshima, Y. 1998. Identification of the human klotho gene and its two transcripts encoding membrane and secreted klotho protein. Biochem. Biophys. Res. Commun. 242:626.[ISI][Medline]
-
Shiraki-Iida, T., Aizawa, H., Matsumura, Y., Sekine, S., Iida, A., Anazawa, H., Nagai, R., Kuro-o, M. and Nabeshima, Y. 1998. Structure of the mouse klotho gene and its two transcripts encoding membrane and secreted protein. FEBS. Lett. 424:6.[ISI][Medline]
-
Okada, S., Nakauchi, H., Nagayoshi, K., Nishikawa, S.-I., Miura Y, and Suda, T. 1992. In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells. Blood. 80:3044.[Abstract]
-
Hardy, R. R., Carmack, C. E., Shinton, S. A., Kemp, J. D. and Hayakawa, K. 1991. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173:1213.[Abstract]
-
Horseman, N. D., Zhao, W., Montecino-Rodriguez, E., Tanaka, M., Nakashima, K., Engle, S. J., Smith, F., Markoff, E. and Dorshkind, K. 1997. Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the prolactin gene. EMBO J. 16:6926.[Abstract/Free Full Text]
-
Nakano, T. Kodama, H. and Honjo, T. 1994. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265:1098.[ISI][Medline]
-
Iscove, N. N., Sieber, F. and Winterhalter, K. H. 1974. Erythroid colony formation in cultures of mouse and human bone marrow: analysis of the requirement for erythropoietin by gel filtration and affinity chromatography on agaroseconcanavalin A. J. Cell. Physiol. 83:309.[ISI][Medline]
-
Okada, S., Nakauchi, H., Nagayoshi, K., Nishikawa, S., Nishikawa, S.-I., Miura, Y. and Suda, T. 1991. Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule. Blood 78:1706.[Abstract]
-
Suda, T., Okada, S., Suda, J., Miura, Y., Ito, M., Sudo, T., Hayashi, S., Nishikawa, S.-I. and Nakauchi, H. 1989. A stimulatory effect of recombinant murine interleukin-7 (IL-7) on B-cell colony formation and an inhibitory effect of IL-1 alpha. Blood 74:1936.[Abstract]
-
Till, J. E. and McCulloch, E. A. 1961. A direct measurement of radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14:213.[ISI]
-
Lamar, E. E. and Palmer, E. 1984. Y-encoded, species-specific DNA in mice: evidence that the Y chromosome exists in two polymorphic forms in inbred strains. Cell 37:171.[ISI][Medline]
-
Yonemura, Y., Ku, H., Hirayama, F., Souza, L. M. and Ogawa, M. 1996. Interleukin 3 or interleukin 1 abrogates the reconstituting ability of hematopoietic stem cells. Proc. Natl Acad. Sci. USA 93:4040.[Abstract/Free Full Text]
-
Chen, X., Liu, W., Ambrosino, C., Ruocco, M. R., Poli, V., Romani, L., Quinto, I., Barbieri, S., Holmes, K. L., Venuta, S. and Scala, G. 1997. Impaired generation of bone marrow B lymphocytes in mice deficient in C/EBPbeta. Blood 90:156.[Abstract/Free Full Text]
-
Yoder, M. C., King, B., Hiatt, K. and Williams, D. A. 1995. Murine embryonic yolk sac cells promote in vitro proliferation of bone marrow high proliferative potential colony-forming cells. Blood 86:1322.[Abstract/Free Full Text]
-
Yamamoto, H., Hatano, M., Iitsuka, Y., Mahyar, N. S., Yamamoto, M. and Tokuhisa, T. 1995. Two forms of Hox11, a T cell leukemia oncogene, are expressed in fetal spleen but not in primary lymphocytes. Mol. Immunol. 32:1177.[ISI][Medline]
-
Miyaura, C., Onoe, Y., Inada, M., Maki, K., Ikuta, K., Ito, M. and Suda, T. 1997. Increased B-lymphopoiesis by interleukin 7 induced bone loss in mice with intact ovarian function: similarity to estrogen deficiency. Proc. Natl Acad. Sci. USA 94:9360.[Abstract/Free Full Text]
-
Marshall, A. J., Fleming, H. E., Wu, G. E. and Paige, C. J. 1998. Modulation of the IL-7 dose-response threshold during Pro-B cell differentiation is dependent on Pre-B cell receptor expression. J. Immunol. 161:6038.[Abstract/Free Full Text]
-
Danska, J. S., Pflumio, F., Williams, C. J., Huner, O., Dick, J. E. and Guidos, C. J. 1994. Rescue of T cell-specific V(D)J recombination in SCID mice by DNA-damaging agents. Science 266:450.[ISI][Medline]
-
Yasunaga, M., Wang, F., Kunisada, T., Nishikawa, S. and Nishikawa, S.-I. 1995. Cell cycle control of c-kit+ IL-7R+ B precursor cells by two distinct signals derived from IL-7 receptor and c-kit in a fully defined medium. J. Exp. Med. 182:315.[Abstract]
-
Kincade, P.W. 1994. B lymphopoiesis: global factors, local control. Proc. Natl Acad. Sci. USA 91:2888.[Free Full Text]
-
Clark, B. R. and Keating, A. 1995. Biology of bone marrow stroma. Ann. NY Acad. Sci. 770:70.[Abstract]
-
Mayani, H., Guilbert, L. J. and Janowska-Wieczorek, A. 1992. Biology of the hemopoietic microenvironment. Eur. J. Haematol. 49:225.[ISI][Medline]
-
Taichman, R. S. and Emerson, S. G. 1998. The role of osteoblasts in the hematopoietic microenvironment. Stem Cells 16:7.[Abstract/Free Full Text]
-
Okada, S., Wang, Z. Q., Grigoriadis, A. E., Wagner, E. F. and von Ruden, T. 1994. Mice lacking c-fos have normal hematopoietic stem cells but exhibit altered B-cell differentiation due to an impaired bone marrow environment. Mol. Cell. Biol. 14:382.[Abstract]
-
Manolagas, S. C. and Jilka, R. L. 1995. Bone marrow, cytokines, and bone remodeling. Emerging insights into the pathophysiology of osteoporosis. N. Engl. J. Med. 332:305.[Free Full Text]
-
Naman, A. E., Lupton, S., Hjerrild, K., Wignall, J., Mochizuki, D. Y., Schmierer, A., Mosley, B., March, C. J., Urdal, D. and Grillis, S. 1988. Stimulation of B-cell progenitors by cloned murine interleukin-7. Nature 333:571.[ISI][Medline]
-
Hayashi, S., Kunisada, T., Ogawa, M., Sudo, T., Kodama, H., Suda, T., Nishikawa, S. and Nishikawa, S.-I. 1990. Stepwise progression of B lineage differentiation supported by interleukin 7 and other stromal cell molecules. J. Exp. Med. 171:1683.[Abstract]
-
Sudo, T., Ito, M., Ogawa, Y., Iizuka, M., Kodama, H., Kunisada, T., Hayashi, S., Ogawa, M., Sakai, K. and Nishikawa, S.-I. 1989. Interleukin 7 production and function in stromal cell-dependent B cell development. J. Exp. Med. 170:333.[Abstract]
-
Welch, P. A., Burrows, P. D., Namen, A., Gillis, S. and Cooper M. D. 1990. Bone marrow stromal cells and interleukin-7 induce coordinate expression of the BP-1/6C3 antigen and pre-B cell growth. Int. Immunol. 2:697.[ISI][Medline]
-
Nagasawa, T., Kikutani, H. and Kishimoto, T. 1994. Molecular cloning and structure of a pre-B cell growth stimulating factor. Proc. Natl Acad. Sci. USA 91:2305.[Abstract]
-
Kaisho, T., Ishikawa, J., Oritani, K., Inazawa, J., Tomizuka, H., Muraoka, O., Ochi, T. and Hirao, T. 1994. A novel surface molecule of bone marrow stromal cells that supports pre-B cell growth. Proc. Natl Acad. Sci. USA 91:5325.[Abstract]
-
Kincade, P. W., He, Q., Ishihara, K., Miyake, K., Lesley, J. and Hyman, R. 1993. CD44 and other cell interaction molecules contributing to B lymphopoiesis. Curr. Top. Microbiol. Immunol. 184:215.[Medline]
-
Eaves, C. J., Cashman, J. D., Kay, R. J., Dougherty, G. J., Otsuka, T., Gaboury, L. A., Hodde, D. E., Lansdorp, P. M., Eaves, A. C. and Humphries, R. K. 1991. Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow culture. II. Analysis of positive and negative regulators produced by stromal cells within the adherent layer. Blood 78:110.[Abstract]
-
Wang, J., Lin, Q., Langston, H. and Cooper, M.D. 1995. Resident bone marrow macrophages produce type 1 interferons that can selectively inhibit interleukin-7-driven growth of B lineage cells. Immunity 3:475.[ISI][Medline]
-
Jacobsen, K. and Osmond, D. G. 1990. Microenvironmental organization and stromal cell associations of B lymphocyte precursor cells in mouse bone marrow. Eur. J. Immunol. 20:2395.[ISI][Medline]
-
Foster, M., Montecino-Rodriguez, E., Clark, R. and Dorshkind, K. 1998. Regulation of B and T cell development by anterior pituitary hormones. Cell. Mol. Life. Sci. 54:1076.[ISI][Medline]
-
Kooijman, R., Hooghe-Peters, E. H. and Hooghe, R. 1996. Prolactin, growth hormone, and insulin-like growth factor-1 in the immune system. Adv. Immunol. 63:377.[ISI][Medline]
-
Chrousos, G. P. 1995. The hypothalamicpituitaryadrenal axis and immune-mediated inflammation. N. Engl. J. Med. 332:1531.
-
Montecino-Rodriguez, E., Clark, R. G., Powell-Braxton, L. and Dorshkind, K. 1997. Primary B cell development is impaired in mice with defects of the pituitary/thyroid axis. J. Immunol. 159:2712.[Abstract]
-
Montecino-Rodriguez, E., Clark, R. G., Johnson, A., Collins, L. and Dorshkind, K. 1996. Defective B cell development in Snell dwarf (dw/dw) mice can be corrected by thyroxine treatment. J. Immunol. 157:3334.[Abstract]
-
Rothstein, G. 1993. hematopoiesis in the aged: a model of hematopoietic dysregulation. Blood 82:2601.[ISI][Medline]
-
Van Zant, G., de Haan, G. and Rich, I. N. 1997. Alternatives to stem cell renewal from a developmental viewpoint. Exp. Hematol. 25:187.[Medline]
-
Albright, J. W. and Makinodan, T. 1976. Decline in the growth potential of spleen-colonizing bone marrow stem cells of long-lived aging mice. J. Exp. Med. 144:1204.[Abstract]
-
Harrison, D. E. 1983. Long-term erythropoietic repopulating ability of old, young, and fetal stem cells. J. Exp. Med. 157:1496.[Abstract]
-
Harrison, D. E., Astle, C. M. and Lerner, C. 1984. Ultimate erythropoietic repopulating abilities of fetal, young adult, and old adult cells compared using repeated irradiation. J. Exp. Med. 160:759.[Abstract]
-
De Haan, G., Nijhof, W. and Van Zant, G. 1997. Mouse strain-dependent changes in frequency and proliferation of hematopoietic stem cells during aging: correlation between lifespan and cycling activity. Blood 89:1543.[Abstract/Free Full Text]
-
Morrison, S. J., Wandycz, A. M., Akashi, K., Globerson, A. and Weissman, I. L. 1996. The aging of hematopoietic stem cells. Nat. Med. 2:1011.[ISI][Medline]