Rapamycin, but not resveratrol or simvastatin, extends life span of genetically heterogeneous mice.

Rapamycin was administered in food to genetically heterogeneous mice from the age of 9 months and produced significant increases in life span, including maximum life span, at each of three test sites. Median survival was extended by an average of 10% in males and 18% in females. Rapamycin attenuated age-associated decline in spontaneous activity in males but not in females. Causes of death were similar in control and rapamycin-treated mice. Resveratrol (at 300 and 1200 ppm food) and simvastatin (12 and 120 ppm) did not have significant effects on survival in male or female mice. Further evaluation of rapamycin's effects on mice is likely to help delineate the role of the mammalian target of rapamycin complexes in the regulation of aging rate and age-dependent diseases and may help to guide a search for drugs that retard some or all of the diseases of aging.

Genetic regulation of hematopoietic stem cell exhaustion during development and growth.

OBJECTIVE: During aging, hematopoietic stem cell (HSC) exhaustion is more severe in BALB/cByJ (BALB) mice than in C57BL/6J (B6) mice. Our objective is to determine whether HSC exhaustion during development from fetus to adult also is more severe for BALB than for B6 mice. MATERIALS AND METHODS: Hematopoietic stem cells from fetal liver cells (FLCs) and from young adult bone marrow cells (BMCs) were compared using the competitive repopulation assay to measure long-term repopulating ability (LTRA) and HSC expansion after serial transplantation. LTRAs were measured in repopulating units (RU), as the ability to produce donor-type erythrocytes and lymphocytes in lethally irradiated recipients relative to the congenic fresh marrow competitor. To test expansion, FLCs or BMCs were serially transplanted into lethally irradiated carriers whose marrow cells were compared using fluorescence-activated cell staining (FACS), and subsequently tested for LTRA. RESULTS: BALB and B6 FLCs, respectively, repopulated 2.6 and 13.5 times as well as BMCs. LTRAs correlated with HSC expansion for BALB, but not B6. Per million donor cells, CD34(-) HSC-enriched fractions (HEFs) and total RU values were 6.8 and 4.6 times higher for FLCs than for BMCs in BALB carriers, while these ratios were only 1.2 and 0.97 higher in B6 carriers. CONCLUSION: In B6 HSC development, LTRA is dissociated from expansion. Although 1 x 10(6) BMCs have much lower LTRA, they expand HSCs as well as 1 x 10(6) FLCs. HSC expansion is partly exhausted in BALB, but not B6, during development.

Hematopoietic stem cell functional failure in interleukin-2-deficient mice.

The effects of interleukin-2 (IL-2) deficiency on hematopoiesis were tested by measuring cellular compositions in peripheral blood, spleen, thymus, and bone marrow of 3- to 5-month-old gene-targeted Il2 null (Il2(-/-)) mice using the Advia 120 Hematology system and fluorescence-activated cell staining (FACS). Il2(-/-) mice developed hematological failure and autoimmune responses, showing variable but significant degrees of anemia, lymphocytopenia, thrombocytopenia, splenomegaly, thymus involution, and weight loss. Surprisingly, Il2(-/-) mice had normal numbers of bone marrow cells (BMCs) with increased numbers of Lin(-)Kit(+)Sca1(+)CD34(-) and Lin(-)Kit(+)Sca1(+)CD34(+) cells that are normally associated with hematopoietic stem cells (HSCs) and progenitor cells. Day-12 colony-forming units-spleen cells were slightly reduced in Il2(-/-) mice. When Il2(-/-) and Il2(+/+) mice were compared for long-term HSC function in vivo in the competitive repopulation assay, BMCs from Il2(-/-) donors had 10- to 20-fold less HSC repopulating ability, which affected both myeloid and lymphoid cell lineages. Thus, HSCs from Il2(-/-) mice can proliferate normally but are functionally defective for reconstituting lethally irradiated recipients.

Maximum life spans in mice are extended by wild strain alleles.

The genes that control basic aging mechanisms in mammals are unknown. By using two four-way crosses, each including a strain derived from wild, undomesticated stocks, we identified two quantitative trait loci that extend murine life spans by approximately 10%. In one cross, the longest-lived 18% of carriers of the D8Mit171 marker allele from the MOLD/Rk strain, Mus m. molossinus, outlived the longest lived 18% of noncarriers by 129 days (P = 5.4 x 10(-5)); in a second cross, carriers of the D10Mit267 allele from the CAST/Ei strain, Mus m. castaneus, outlived noncarriers by 125 days ( P = 1.6 x 10(-6)). In both crosses, P < 1.0 x 10(-4 )is considered significant. Because these life span increases required that all essential biological systems function longer than normal, these alleles most likely retarded basic aging mechanisms in multiple biological systems simultaneously.

Analysis of the stem cell sparing properties of cyclophosphamide.

OBJECTIVES: Cyclophosphamide was examined for its ability to spare the most primitive hematopoietic stem cell (PHSC). METHODS: C57BL6/J mice (Groups A and B) were sacrificed 24 h and 4-6 wk, respectively, after a single or second injection of low-dose cyclophosphamide (90 mg/kg) on days 1, 3, 7, or 15. A competitive repopulation assay was then performed, using B6-HbbdGpi-1a competitor cells, to determine the repopulating ability of exposed PHSC. RESULTS AND CONCLUSIONS: PHSC function was preserved after a single injection of cyclophosphamide and after a second injection on days 7 and 15 in both groups. In Group A, PHSC repopulating ability declined after a second injection on days 1 and 3 (p<0.05 only for day 1), as did repopulating units [RU]; PHSC numbers did not. In Group B, an insignificant decrease in repopulating ability and RU numbers was observed after a second injection on days 1 and 3, suggesting different etiologies for losses in the 2 groups, or correction of drug-induced defects within 1 month of cyclophosphamide administration. Total RU increased in single, day 1, 7 and 15 treatment groups. A significant number of marrow cells entered the S phase after cyclophosphamide dosing on day 3, and it is possible that a relationship exists between cell cycling and replicative damage. DNA damage was also increased 1 and 3 d after cyclophosphamide administration, although the significance of differences from controls was not definitive. CONCLUSION: Low-dose cyclophosphamide can spare stem cells, depending upon the timing of subsequent doses.

Primitive hematopoietic stem cell function in vivo is uniquely high in the CXB-12 mouse strain.

Bone marrow cells (BMCs) from CXB-12/HiaJ (CXB-12) mice had 14 times the total long-term repopulating ability found in the best of 11 other CXB recombinant inbred (RI) lines. BMCs from each RI line donor were mixed with genetically marked standard competitor BMCs from the BALB/cByxC57BL/6 F1 (CByB6F1) hybrid, the mice used to produce the RI lines, and the mixtures repopulated lethally irradiated CByB6F1 recipients. Percentages of donor-type erythrocytes and lymphocytes measured the actual long-term repopulating functions of the donor RI lines relative to the standard competitor. CXB-12 BMCs repopulated better after 3 or 6 months than after 1 month, suggesting that the most primitive precursors were involved. Compared to CByB6F1 standard competitor cells, CXB-12 cells repopulated 3 to 12 times as well, with their advantage increasing when higher doses of cells were transplanted, probably because of hybrid resistance of the recipient against low doses. This was far better than expected, because F1 cells normally function 2 to 3 times as well as cells from an inbred strain. In competitive dilution, the advantage resulted from 2 factors: more precursor cells and more function per precursor. In the model that best fit the data, CXB-12 donors had 2.4 times the concentration of hematopoietic stem cells (HSCs) as the CByB6F1 standard, and each HSC repopulated 1.4 times as well. CXB-12 mice did not have elevated erythrocyte and lymphocyte numbers in blood and marrow and did not have unusually elevated concentrations of colony-forming unit spleen, cobblestone colonies, and long-term colony-initiating cells in marrow.

Genetic regulation of primitive hematopoietic stem cell senescence.

OBJECTIVE: To define effects of strain on PHSC (primitive hematopoietic stem cells) senescence (decline in function with age) in vivo, and to map a locus that regulates PHSC senescence. MATERIALS AND METHODS: Long-term function and self-renewal were compared in bone marrow cells (BMC) from old and young mice of three strains: BALB/cBy (BALB), DBA/2 (D2) and C57BL/6 (B6), using competitive repopulation and serial transplantation in vivo. BMC from each old or young donor were mixed with standard doses of congenic, genetically marked BMC and transplanted into lethally recipients. Percentages of donor-type erythrocytes and lymphocytes in the recipients determined the functional ability of donor PHSC relative to the standard, where one repopulating unit (RU) of donor BMC equals the repopulating ability of 100,000 standard competitor BMC. Using similar techniques, repopulating abilities of old and young recombinant inbred (RI) donors of 12 strains derived from BALB and B6 were compared in NK-depleted BALBxB6 Fl recipients to map a locus that appears to have a major role in PHSC senescence. RESULTS: PHSC function declined about 2 fold with age in BALB and D2 BMC, and increased more than 2-fold with age in B6 BMC, with all old/young strain differences significant, p<.01. Ten months after serial transplantation, young B6, BALB, and D2 PHSC had self-renewed 1.6-, 4.2-, and 3.2-fold better than old, with BALB and D2 old/young differences p<.01. Young B6 PHSC self-renewed 1.9- and 2.9-fold better than young BALB and D2 PHSC. The PHSC senescence phenotypes (old/young RU ratios) for 12 CXB RI strains suggested a genetic linkage to D12Nyul7 on Chromosome 12. CONCLUSION: PHSC senescence is genetically regulated, and is much delayed in the B6 strain compared to the BALB and D2 strains. A locus on Chromosome 12 may regulate PHSC senescence.

Development and aging of primitive hematopoietic stem cells in BALB/cBy mice.

Evaluating the function of an individual hematopoietic stem cell (HSC) is a difficult and important problem. The functional ability per HSC, as well as the HSC concentration, was measured with minimal disruption to the cells in vivo using the new competitive dilution assay. Distribution of HSC into recipients was modeled based on Poisson probabilities. Predictions of donor contributions from models assuming different levels of donor HSC functional ability and concentration were compared to actual observations. The model with the least difference between predictions and observations was accepted. In BALB/ cBy (BALB) mice, models assuming equal functional ability of HSC from the same donor fit extremely well with actual observations, suggesting that all HSC are functionally homogeneous at any particular time point during development or aging. Relative HSC functional ability per cell declined during development, so that a fetal HSC had 1.6 to 3.0 times the functional ability of a young adult HSC. The decline continued with age, so that a young adult HSC had 1.6 to 2.0 times the functional ability of an old HSC. Concentrations of HSC that engrafted and functioned were similar among 16-day fetal liver cells and bone marrow cells (BMC) from 3-month and 25 to 28-month-old adult mice. They were either 10 or 4 HSC per million cells when tested in BALB or CByB6F1 recipients, respectively. All HSC were pluripotent and produced lymphoid and myeloid descendants proportionally (r = 0.80 to 0.98, p < 0.01). Fetal and young HSC in both types of recipients maintained clonal stability long term so that percentages of donor cells at 6 and 9 months were strongly correlated (r = 0.72 to 0.93, p < 0.01). Although HSC from aged donors in BALB recipients maintained clonal stability, HSC from the same aged donors failed to show clonal stability in CByB6F1 recipients, perhaps due to the less suitable host environment. All HSC from BALB mice seemed to have equal functional levels at a given stage of life and were gradually exhausted simultaneously through development and aging.

Delayed immune aging in diet-restricted B6CBAT6 F1 mice is associated with preservation of naive T cells.

Age-related changes in peripheral blood, spleen, and thymus of ad libitum (AL)-fed and dietary restricted (DR) C57BL/6J x CBA/CaH-T6/J F1 (B6CBAT6 F1) mice at young (3 mo), middle (16 mo), and old (30 mo) ages were studied to define how dietary restriction retards immune aging. Dietary restriction at 25% AL intake level initiated at weaning significantly reduced the rates of age-related declines in peripheral blood T helper cells, naive T helper cells, and naive cytotoxic T lymphocytes (CTLs). As a result, concentrations of these cell types in old DR mice were equivalent to 161%, 176%, and 250% of those in old AL controls. Dietary restriction also abolished age-related splenomegaly and decreased total splenocyte numbers in old DR mice. Dietary restriction did not prevent age-related decline in thymus size, but preserved thymus cellularity in old mice. Old DR mice had twice as many total thymocytes and 2.6 times as many CD4+CD8+ immature thymocytes as old AL controls. The correlations between total immature thymocytes and concentrations of circulating naive T helper cells and naive CTLs increase with age and become significant in old mice. Thus, dietary restriction preserves immature T-cell precursors in the thymus during aging to maintain higher concentrations of circulating T helper and naive T cells in peripheral blood.

Stem cell factor improves the repopulating ability of primitive hematopoietic stem cells after sublethal irradiation (and, to a lesser extent) after bone marrow transplantation in mice.

Bone marrow transplantation (BMT) and sublethal irradiation (XRT) cause profound long-term damage to hematopoietic stem cells. We used the competitive repopulation assay in mice to test the ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF), cytokines given in clinical settings to enhance marrow recovery after XRT or BMT and to protect the marrow repopulating ability of primitive hematopoietic stem cells (PHSC) after these modalities. The repopulating ability of exhaustible multilineage progenitors (EMP) was also tested after these modalities, with or without cytokines. Repopulating abilities of EMP and PHSC were significantly reduced after XRT or BMT; PHSC were preferentially affected. Administration of SCF to C57B6/J mice after XRT resulted in improved EMP and PHSC repopulating ability, although progenitor numbers--repopulating units--were not completely returned to control levels. Whether given as a single dose or multiple doses, GM after XRT did protect PHSC function from the deleterious effects of XRT, but this was not a significant effect. SCF caused an increase in PHSC repopulating ability after BMT, but this too was not a significant difference. GM after BMT had little effect. SCF administration before XRT led to severe impairment of PHSC function with very little or no stem cell activity observed. Therefore, timing of its administration is an important consideration since preadministration of the cytokine before XRT can be extremely harmful to PHSC function.

Short- and long-term multilineage repopulating hematopoietic stem cells in late fetal and newborn mice: models for human umbilical cord blood.

Blood from late fetal and newborn mice is similar to umbilical cord blood obtained at birth in human beings, an important source of stem cells for clinical transplantation. The mouse model is useful because long-term functions can be readily assayed in vivo. To evaluate the functions of hematopoietic precursors in the blood and other tissues of late fetal and newborn mice, short- and long-term multilineage repopulating abilities were measured in vivo by competitive repopulation. Manipulations that might affect cell function, such as enrichment, tissue culture, or retroviral marking, were avoided. Hematopoietic stem cell functions of late fetal or newborn blood, liver, and spleen, were assayed as myeloid and lymphoid repopulating abilities relative to standard adult marrow cells. Donor cells from these tissues as well as adult control donor marrow cells were all of the same genotype. Cells from each donor tissue were mixed with portions from a pool of standard adult "competitor" marrow distinguished from the donors by genetic differences in hemoglobin and glucosephosphate isomerase. After 21 to 413 days, percentages of donor type myeloid and lymphoid cells in recipient blood were measured to assay the functional abilities of donor precursors relative to the standard. These relative measures are expressed as repopulating units, where each unit is equivalent to the repopulating ability found in 100,000 standard adult marrow cells. Thus, measures of repopulating units do not compare single cells but overall repopulating abilities of donor cell populations. Relative functional abilities in 1 million nucleated cells from late fetal or newborn blood were several times less than those found in adult marrow, but far more than in normal adult blood, and appeared to include long-term functional primitive hematopoietic stem cells (PHSC) similar to those in marrow. To estimate functional abilities of individual PHSC, variances among large groups of identical recipients were analyzed using both the binomial model and competitive dilution, a new model based on the Poisson distribution. The data best fit the hypothesis that individual PHSC from adult marrow, late fetal blood, or newborn blood each produce similar fractions of the total lymphoid and erythroid cells found in the recipient for many months.

Hematopoietic precursor cell exhaustion is a cause of proliferative defect in primitive hematopoietic stem cells (PHSC) after chemotherapy.

The authors used the competitive repopulation assay and simple statistical analyses to estimate concentrations of primitive hematopoietic stem cells (PHSCs) in the marrow of mice after chemotherapy. Single doses of cyclophosphamide (CTX) from 80 to 200 mg/kg were administered to C57B16/J mice. Other treatment groups included mice given multiple doses of CTX at the lowest dose of 80 mg/kg; mice given four weekly doses of vincristine (VCR) or vinblastine (VBL); mice given two biweekly doses of bleomycin; mice receiving cytosine arabinoside (ARA) administered intraperitoneally thrice daily or as a continuous infusion by Alzet pump for 3 days; and controls given no drug. The lowest dose of CTX (80 mg/kg), given once or repeatedly, spared PHSC numbers and function. The functional capacity of PHSCs declined significantly once doses of CTX exceeded 100 mg/kg. Decreases in PHSC function were usually associated with reductions in PHSC numbers; repopulating units, which include all repopulating cells, were similarly reduced. At the highest dose (33 mg/kg for 3 days), ARA caused a decline in marrow repopulating function. Drugs associated with mild clinical myelosuppression, such as VCR and VBI, did not significantly affect the repopulating ability of PHSCs, although VCR caused drastic declines in PHSC numbers. The marrow reconstitutive defects clinically-observed after chemotherapy may be caused partly by depletion of the PHSC pool.

Relative to adult marrow, fetal liver repopulates nearly five times more effectively long-term than short-term.

Multilineage precursor cells from 14-day B6 (C57B1/6J) mouse fetal liver and adult bone marrow that repopulate both the lymphoid and myeloid systems were compared by competitive repopulation. Cells were assayed in normally functioning populations, and enrichment, tissue culture, and induced marking were avoided since these manipulations might affect cell function. Fetal or adult donor cells were mixed with marked adult competitor cells and transplanted into irradiated recipients whose blood was tested at short (25-33-day) or long (105-245-day) time periods after transplantation. Proportions of lymphocytes, granulocytes, and platelets descended from donor precursors were measured by GPI (glucosephosphate isomerase) isozyme genetic markers in congenic mice, and represent the repopulating abilities of these precursors relative to the standard competitor. For short-term repopulation 25-33 days after transplantation, fetal and adult donor cells were similar; in three studies, fetal liver contributed 0.8, 1.1, and 1.4 times as much as adult marrow per 10(5) cells transplanted. However, when long-term (105-245-day) repopulation was tested in the same recipients, fetal liver contributed 3.5, 5.0, and 7.1 times as much as adult marrow. Ratios of long-term/short-term repopulating abilities in fetal liver relative to standard adult marrow competitors were 2.5, 8.9, and 4.7, while in marrow controls, these ratios remained approximately one (1.14 and 0.80). Thus, 14-day fetal liver contains several times more long-term repopulating cells relative to short-term repopulating cells than does adult marrow. Ratios of long-term/short-term fetal cells were unchanged by precursor enrichment. The AA4.1+, Ly-6A/E+, lineage low fraction had a ratio of 4.4, although it repopulated 276 times better than unenriched fetal cells whose ratio was 4.7. There are two hypotheses that explain these data most simply: 1) There may be only a single multilineage precursor, but after transplantation cells seed in different microenvironments that support either long-term or short-term function. 2) Conversely, the difference may be at the stem cell level rather than the microenvironmental level, so that there are tow types of stem cells with multilineage differentiating ability, but only one functions over the long-term. The current report defines new conditions required by each hypothesis. If functional life spans are defined by seeding sites, as in hypothesis 1, fetal cells seed much higher proportions of long-term sites than adult cells. If different types of stem cells function short-term and long-term, as in hypothesis 2, they are not distinguished by markers allowing a 276-fold enrichment to 1367 times the repopulating ability of fresh marrow.

Distinct developmental patterns of short-term and long-term functioning lymphoid and myeloid precursors defined by competitive limiting dilution analysis in vivo.

Functional abilities of individual marrow precursor cells were defined by competitive limiting dilution without enrichment, tissue culture, or induced marking, manipulations that might affect cell functions. We directly measured long-term repopulating abilities in limiting doses (0.25-1.0 x 10(5)) of genetically marked congenic marrow cells. These were mixed with a standard dose of 4 or 5 x 10(5) competitor marrow cells, which contained a predictable distribution of precursor cells and allowed quantitative assays. Percentages of donor type T and B lymphocytes, granulocytes, platelets, and erythrocytes were measured in recipient blood. Applying the maximum likelihood statistic, concentrations (per 10(5)) of precursors repopulating at least one lineage were: 4.7 and 6.0 after 6 wk, 1.6 and 2.7 after 14 to 15 wk, and 1.2 and 1.9 after 30 to 32 wk; concentrations repopulating at least three lineages were 2.3 and 3.4 after 6 wk, 0.9 and 1.7 after 14 to 15 wk, and 0.9 and 1.3 after 32 wk. Almost all precursors functioning after 14 wk repopulated all lineages. At 6 wk, similar levels of donor cells were produced in recipients of both short- and long-term precursors. However, after 14 to 32 wk, contributions by short-term precursors (about two-thirds of the precursors) dropped to zero, while contributions by long-term precursors (about one-quarter of the precursors) expanded severalfold. The latter permanently repopulated all lineages after 30 to 32 wk, functioning as the most primitive stem cells (PSC) in the immune and myeloid systems. Nearly all the variance in long-term repopulated recipients was explained using the Poisson distribution to calculate donor percentages in a model where each donor and competitor PSC contributed equally.

Long-term repopulating abilities of enriched fetal liver stem cells measured by competitive repopulation.

To characterize hematopoietic cell biology, many investigators have used protocols that enrich for primitive hematopoietic stem cells (PHSC). In this study, we quantified the long-term repopulating ability (LTRA) of enriched and discarded fractions of PHSC from day-14 murine fetal liver using the competitive repopulation assay. We fractionated populations of fetal cells using the antigenic markers AA4.1+, AA4.1+/Sca+, and AA4.1+/Linlow/Sca+. Differentiating and repopulating abilities of each of these populations were directly compared using competitive repopulation. Adult bone marrow was mixed with fetal cell fractions from congenic donors having genetically distinguishable markers, and mixtures were given to irradiated recipients. Differentiating and repopulating abilities of the enriched donor cells were measured by the proportions of myeloid and lymphoid cells having donor markers that repopulated the recipients. LTRA was found primarily in the AA4.1+ and AA4.1+/Sca+ subpopulations. Further fractionation of the AA4.1+ cells to derive an AA4.1+/Linlow/Sca+ fraction showed that virtually all of the long-term stem cell activity was found in this subpopulation. These cells were 1400- to 1600-fold enriched in long-term functional ability compared to fresh marrow. This very high multilineage repopulating ability per cell was directly measured using a long-term functional assay in vivo. Importantly, the measured repopulating ability for AA4.1+/Linlow/Sca+ cells was about five-fold less than expected from the fraction of cells enriched and remained two- to three-fold less even after compensating for repopulating ability in discarded fractions. This illustrates that long-term functional abilities of enriched PHSC cannot be estimated from fractions enriched but should be quantitatively assayed.

Splenic primitive hematopoietic stem cell (PHSC) activity is enhanced by steel factor because of PHSC proliferation.

To test whether primitive hematopoietic stem cells (PHSCs) are stimulated by Steel (SI) factor (c-kit ligand) in vivo, donor mice were studied after three or seven daily injections of SI factor. PHSC activity was measured as long-term erythroid and lymphoid competitive repopulating ability. Cells to be tested (usually marrow or spleen cells from treated donors) were mixed with untreated competitor marrow that produces erythrocytes and lymphocytes that are genetically distinguishable from the donors by differences in hemoglobin (Hb) and glucosephosphate isomerase (GPI) markers. These cell mixtures were injected into lethally irradiated hosts, and after 111 to 293 days, functional abilities of donor PHSC populations were assessed and expressed as percentages of donor-type Hb and GPI in the host's circulating erythrocytes and lymphocytes, respectively. A striking increase in splenic PHSC activity occurred after seven daily injections of SI factor, with a much smaller increase after three daily injections. Both three and seven daily injections of SI factor slightly reduced marrow PHSC activity. Rapid cycling greatly increases PHSC vulnerability to 5-fluorouracil (5FU). To test whether SI factor stimulates PHSCs into rapid cycling, donor mice were given a dose of 5FU in addition to SI factor. The increase in splenic PHSCs after 7 days of treatment with SI factor occurred to a similar degree whether donors were or were not treated with 5FU on day 8. However, a dose of 5FU on day 4 of the SI factor treatments almost totally prevented the increase in splenic PHSC activity. Apparently this increased activity requires PHSC cycling throughout the period of SI factor treatment.

Primitive hemopoietic stem cells: direct assay of most productive populations by competitive repopulation with simple binomial, correlation and covariance calculations.

Quantitative analyses of primitive hemopoietic stem cell (PHSC) populations are important both for basic biology and for clinical applications. Unfortunately, many conventional assays fail to measure long-term repopulating ability and maximal differentiating ability, the most important characteristics of the PHSC. The competitive repopulation assay described here focuses on this characteristic, assaying the precursors from which most differentiated cells are descended over large fractions of the life span in laboratory mice. Thus long-term repopulating ability and the ability to differentiate into both myeloid and lymphoid lineages are measured directly from 2.5 to 12.5 months after transplantation. This technique also has found high correlations between granulocytes, macrophages, and T and B lymphocytes as early as 3 weeks after transplantation. All or most differentiated cells of these widely disparate types appear to be descended from a common precursor cell, while myeloid-specific or lymphoid-specific precursors produce few or no descendants. However, large increases in variances between 3 and 6 weeks and 12 weeks after transplantation suggest that most of the initially active multilineage precursors are exhausted. Thus the ability to differentiate into widely disparate lineages does not establish long-term repopulating ability.

Assessing permanent damage to primitive hematopoietic stem cells after chemotherapy using the competitive repopulation assay.

The competitive repopulation assay was used to document the effects of six chemotherapeutic agents on primitive hematopoietic stem cells. The assay measures the relative abilities of donor cells to produce circulating erythrocytes and lymphocytes in lethally irradiated congeneic mice over a period of 6 months. Long-lasting marrow reconstitutive deficits in cells of donor origin occurred after exposure to 5-fluorouracil (5FU), bis-chloronitrosourea (BCNU), cyclophosphamide (CTX), vincristine (VCR), and actinomycin D (ACT) but not after exposure to cytosine arabinoside (ARA). Repopulating abilities were reduced after as little as a single dose of CTX or BCNU. A second dose of BCNU caused even more severe effects. A single dose of 5FU had no effect on repopulating abilities despite a temporary 10-fold reduction in marrow cell number, but multiple doses reduced the marrow stem-cell replicative ability to less than half of the normal control levels. These effects were not reliably predicted or detected by colony-forming assays or by reductions in marrow cell number. Thus, long-lasting proliferative defects in the primitive hematopoietic stem-cell (PHSC) population can result from the use of chemotherapeutic agents. Such findings may have clinical implications, especially in individuals receiving repeated or prolonged administration of these agents or in instances of marrow transplantation.

Lymphoid and erythroid repopulation in B6 W-anemic mice: a new unirradiated recipient.

The W-anemic family of mouse mutants is an important model for studying repopulation in unirradiated recipients. This is the first study of blood lymphoid cell repopulation in adult W-anemic mutants given high doses of marrow cells, and it shows a wide difference in repopulation rates of circulating lymphoid and erythroid cells. This study also offers an improved model for marrow transplantation, using W alleles that are spontaneous mutations on the widely used inbred strain C57BL/6J (B6). Unirradiated B6-W41J/W41J or -W41J/W39J recipients of 2 x 10(6) B6 marrow cells are completely repopulated with donor erythrocytes after 3 months, whereas complete repopulation of lymphocytes requires a year. Surprisingly, the eventual degree of repopulation is independent of the severity of the mutation. The new mutants are not as anemic as the commonly used WBB6F1-W/Wv anemic mutants, they have a much higher ability to form macroscopic spleen colonies (spleen colony-forming units, CFU-S), and B6-W41J/W41J mice are fertile. Nevertheless, lymphoid and erythroid repopulation occur to a similar extent in B6-W41J/W41J or -W41J/W39J and in WBB6F1-W/Wv anemic mutants. Repopulation is more rapid in the latter, but host cells may be damaged by B6 reactions against the WB parent. Avoiding graft-versus-host reactions, hybrid resistance, and similar complications are important advantages in using donors and unirradiated recipients all on the B6 mouse genetic background. Additionally, congenic B6 mice provide a variety of genetic markers, allowing myeloid and lymphoid repopulation to be readily quantitated.

Effects of transplantation on the primitive immunohematopoietic stem cell.

Transplantation has strong deleterious effects on the primitive immunohematopoietic stem cells (PSC) from which circulating lymphocytes and erythrocytes are descended. We studied these effects over 300-400 d, testing whether PSC numbers, repopulating abilities, or both, were reduced. Equivalent PSC numbers were estimated in recipients of mixtures of genetically different cells, using the binomial model with covariance. Percentages of lymphocyte and erythrocyte types were closely correlated, as were percentages of either type sampled at intervals of several months. This suggests that the same PSC produced lymphoid and myeloid cells, and that most circulating cells were descended from the same PSC over hundreds of days. Equivalent PSC concentrations were approximately 1/10(5) fresh marrow cells, and were about twofold lower using previously transplanted marrow. However, such marrow repopulated only one-seventh to one-eighth as well as fresh marrow. Apparently, transplantation not only reduces PSC concentrations, but also reduces the repopulating ability per PSC. This may result from excessive stimuli to differentiate that overbalance the stimuli for PSC to replenish themselves.