Defective in vitro migratory capacity of bone marrow cells from viable motheaten mice in response to normal thymus culture supernatants

"Viable motheaten" mice are severely immunodeficient and develop autoantibodies early in life. The thymus appears normal for the first 3-4 weeks, after which there is depletion of cortical thymocytes and a diminution in the size of the organ until it is atrophic. The present study utilized an in vitro migration assay, in which bone marrow cells from viable motheaten mice were found to have a greatly diminished capacity to migrate in response to normal thymus supernatant when compared to normal bone marrow cells. It was also determined that thymus supernatant prepared from newborn viable motheaten mice was chemoattractive to normal bone marrow but not to viable motheaten bone marrow. The results of in vivo reconstitution of lethally irradiated viable motheaten mice with normal bone marrow cells also show that the thymus of the mutant is normal in its ability to attract and be repopulated by normal donor bone marrow. Therefore, the premature thymic involution of viable motheaten mice is related to the inability of bone marrow cells from these mice to migrate or respond to signals from the thymus.     

Comparison of the effectiveness of bone marrow and spleen stem cells for platelet repopulation in lethally irradiated mice

To determine whether stem cells from spleen differ from those of bone marrow in their ability to support platelet repopulation after lethal irradiation we compared normal, splenectomized and plethoric (3 weeks exposure to CO) spleen cell recipients with similar groups of mice reconstituted with comparable numbers of bone marrow stem cells. Female LAF1 mice were given 825-950 R from a 60Co source and were transplanted with 0, 0.5, 1, 2, or 4 x 10(6) bone marrow or 10-64 x 10(6) spleen cells. CFUs content of the transplant was calculated from day 9 splenic nodules in mice receiving 5 x 10(4) bone marrow or 1.0-1.6 x 10(6) spleen cells. Blood volumes, platelet counts and hematocrits were determined on day 12. Total circulating platelets increased with increasing stem cell dose (range 50-1000 CFUs) after both types of transplant. Plethoric mice always had lower platelet levels than controls even when corrected for expanded blood volume. There was little difference between values for normal and splenectomized mice. Platelet production per CFUs in all groups except splenectomized spleen cell recipients declined with large transplants presumably because of feedback inhibition. Although the number of splenic megakaryocytes in spleen cell recipients was approximately 2.5 times as great as in bone marrow recipients, platelet levels were significantly higher only in normal and splenectomized mice receiving more than 300 CFUs. No differences were found between the two types of transplant when plethoric hosts were used.     

Engraftment of bone marrow cells into normal unprepared hosts: effects of 5-fluorouracil and cell cycle status

Bone marrow from animals treated with 5-fluorouracil (5FU) competes equally with normal marrow when assessed in vivo in an irradiated mouse, but shows markedly defective engraftment when transplanted into noncytoablated hosts. Using Southern Blot analysis and a Y-chromosome specific probe, we determined the level of engraftment of male donor cells in the bone marrow, spleen, and thymus of unprepared female hosts. We have confirmed the defective engraftment of marrow harvested 6 days after 5FU (FU-6) and transplanted into unprepared hosts and shown that this defect is transient; by 35 days after 5FU (FU-35), engraftment has returned to levels seen with normal marrow. FU-6 marrow represents an actively cycling population of stem cells, and we hypothesize that the cycle status of the stem cell may relate to its capacity to engraft in the nonirradiated host. Accordingly, we have evaluated the cycle status of engrafting normal and FU-6 marrow into normal hosts using an in vivo hydroxyurea technique. We have shown that those cells engrafting from normal marrow and over 70% of the cells engrafting from FU-6 marrow were quiescent, demonstrating no killing with hydroxyurea. We have also used fluorescent in situ hybridization (FISH) analysis with a Y-chromosome probe and demonstrated that normal and post-5FU engraftment patterns in peripheral blood were similar to those seen in bone marrow, spleen, and thymus. Altogether these data indicate that cells engrafting in normal, unprepared hosts are dormant, and the defect that occurs after 5FU is concomitant with the induction of these cells to transit the cell cycle.     

Thymus regeneration by bone marrow cell suspensions differing in the potential to form early and late spleen colonies

We have determined the thymus-repopulating capacity of purified hemopoietic stem cells, bone marrow cells from mice injected four days previously with 5-fluorouracil (5-FUBM), and bone marrow cells cultured in the presence of stem-cell-activating factor (SAF; SAFBM). SAF is identical to interleukin 3 (IL-3). Purified stem cells are more enriched in day-12 CFU-S than in day-8 CFU-S. 5-FUBM consists of CFU-S that give rise to late (day-12) spleen colonies. SAFBM contains predominantly CFU-S that give rise to early spleen colonies (days 6-8). There is also a net increase in the number of spleen colony-forming units (CFU-S) in these cultures. Thymus regeneration after transplantation with either purified stem cells or 5-FUBM was delayed approximately three days as compared with that after transplantation with normal bone marrow cells. This delay can be ascribed to the absence of prothymocytes in these preparations. Thymus regeneration by SAFBM was delayed approximately ten days as compared with that after transplantation with normal bone marrow cells. The most likely explanation of these results is as follows. The prothymocytes in normal bone marrow produce a relatively limited offspring in the thymus soon after transplantation. This is rapidly replaced by the offspring of newly formed prothymocytes, the results of differentiation of the pluripotent stem cells. These stem cells also give rise to late spleen colonies. Stem cells that give rise to early spleen colonies appear to have lost the capacity for differentiation into the T-cell lineage.     

Hematologic abnormalities of the immunodeficient mouse mutant, viable motheaten (mev)

We have studied the hematopoietic system of the immunodeficient mouse mutant, viable motheaten (mev/mev). These mice usually die by 9 weeks of age from severe pneumonitis. The lungs at that time are infiltrated with granulocytes, macrophages, and lymphocytes. Granulocyte and macrophage precursor cells (CFU-GM) are dramatically increased in the spleens of mev/mev mice, whereas the bone marrow population of these precursors is decreased when compared with littermate control animals. The CFU-GM population retained its normal dependence on granulocyte-macrophage colony-stimulating factor (GM-CSF) for proliferation and differentiation. In contrast, the frequency of an erythroid precursor (CFU-E) was dramatically increased in spleen and showed increased sensitivity to erythropoietin (Epo). Moreover, a splenic CFU-E subpopulation formed normally appearing erythroid colonies in the absence of exogenous Epo. The bone marrow CFU-E population was significantly diminished in size when compared with either wildtype C57BL/6J mice or mice heterozygous for the mev allele. Unlike the CFU-E population, erythroid burst-forming unit (BFU-E) frequency in mev/mev mice was diminished both in bone marrow and in spleen, although the total number of splenic BFU-E was increased because of splenomegaly in these animals. BFU-E retained their dependence on the presence of both Epo and a source of interleukin 3 (IL-3) for proliferation and differentiation into erythroid bursts. Spleen cells from mev/mev mice, when stimulated in vitro with pokeweed mitogen, failed to produce significant quantities of IL-3. Comparison with medium or +/mev heterozygotes revealed that mev/mev spleen cell-conditioned medium showed a 40-fold reduction in burst-promoting activity. Thus, in viable motheaten mice, there is a major shift in hematopoiesis from bone marrow to spleen, which is accompanied by a diminished capacity of spleen cells to produce burst-promoting activity. These data and those from other studies suggest that the hematopoietic microenvironment of marrow may be impaired in this mutant.     

Murine "viable motheaten" mutation reveals a gene critical to the development of both B and T lymphocytes

In lethally irradiated normal mice reconstituted with both normal and autoimmune mutant viable motheaten (mev) bone marrow, the mev-derived B and T cells display aberrant behavior, while those derived from the normal bone marrow develop and function normally. The observed developmental abnormalities of mev B and T lymphocytes are therefore intrinsic to these cell types, rather than being determined by defective influences from the cells' environment. These data bring into question the in vivo significance of reported intercellular regulatory defects in motheaten (me) and mev mice and suggest that these mutations affect a gene whose product acts cell autonomously in the development of several hematopoietic cell lineages including B and T lymphocytes.     

In vivo administration of stem cell factor to mice increases the absolute number of pluripotent hematopoietic stem cells

We have examined the effects of administration of stem cell-factor (SCF) on the number and distribution of pluripotent hematopoietic stem cells (PHSC) in normal mice. Using the competitive repopulation assay we found that in vivo administration of SCF increases the absolute number of PHSC per mouse threefold. The increased numbers of PHSC are found in the peripheral blood and spleen of the SCF-treated animals. The spleen and peripheral blood stem cells completely repopulated the erythroid, myeloid, and lymphoid lineages of irradiated or W/Wv hosts, similar to bone marrow PHSC. PHSC from the peripheral blood of SCF- treated mice have a lineage marker-negative, c-kit-positive phenotype that is indistinguishable from that of bone marrow PHSC. The increase in the absolute number of spleen PHSC is associated with efficient gene transfer to these cells without prior treatment with 5-fluorouracil. This is a US government work. There are no restrictions on its use.     

Development of hematopoietic spleen colonies in nonirradiated genetically normal mice.

The question as to whether prior irradiation or injection of cytotoxic drugs is essential for the development of spleen colonies was examined in genetically normal mice. Mixtures of lymph node and bone marrow cells from C57BL mice were injected into (C57BL X CBA-T6T6) F1 hybrid mice without pretreatment. Hematopoietic nodules were observed in the spleens of F1 hybrid mice killed 18 days after injection. The average number of nodules increased linearly with increased numbers of injected bone marrow cells. Hematopoietic stem cells (CFU-S) and dividing cells in the nodules were shown to be of C57BL origin. Histologic examination showed that erythroid cell colonies predominated over granulocytic cell colonies. These results suggest that any kind of treatment that causes the depletion of CFU-S in the spleen of hosts would provide a suitable environment for the production of colonies by transplanted CFU-S.     

Hematopoiesis of hereditarily asplenic-athymic (lasat) mice.

The hematopoiesis of athymic-asplenic (lasat) mice was compared with that of normal, asplenic, and athymic littermates with the same strain background. Erythrocyte blood volume, number and survival time were normal when related to the body weight of the animals. Peripheral blood showed leukopenia with absolute and relative lymphopenia, resembling the athymic rather than the asplenic pattern. The bone marrow was hypocellular as a consequence of a decrease in both lymphocytes and erythroid precursors, while thrombocytopoiesis and granulcytopoiesis-monocytopoiesis were essentially normal. Although the percentile value of femoral stem cells was high, their absolute number was, in fact, reduced by 35% as a result of the bone marrow hypocellularity. When lasat bone marrow cells were injected into normal, lethally irradiated mice, a rapid erythropoietic recovery was observed, whereas the restoration of the granlocytic compartment was impaired. It was concluded that: 1) lasat mice depict a normal hematopoiesis in spite of the congenital absence of the thymus and the spleen; 2) bone marrow stem cells may be defective when administered to lethally irradiated hosts; and 3) the athymic status predominates over the asplenic one.     

A Difference Between Spleen-derived and Bone Marrow-derived Colony-forming Units in Ability to Protect Lethally Irradiated Mice

Suspensions of cells in the bone marrowand in the spleen of normal mice wereprepared. Two groups of lethally irradiated mice were given cells from bonemarrow and two groups of lethally irradiated mice were given cells from thespleen of the same donor mouse. One ofthe groups injected with bone marrowand one injected with spleen cells werekilled at 9 days for determination of CFU.The remaining animals were followedfor 100 days to determine the mortalitystatistics. The mortality among mice notgiven cells was 100 per cent; amongmice injected with cells, the mortalitywas reduced. The data permit a comparison of the reduction in mortality (protection) following injection of bonemarrow CFU with protection followinginjection of spleen CFU. Protection withCFU of bone marrow was different fromprotection with CFU of spleen. It seemspossible that both hemopoietic pluripotent "stem cells" and "early differentiated progenitors" can form spleen colonies and that the ratio of these CFU maybe different for bone marrow and spleen.

Submitted on April 27, 1970 Revised on June 6, 1970 Accepted on June 12, 1970     

Transplantation of chromosomally marked syngeneic marrow cells into mice not subjected to hematopoietic stem cell depletion

The percentage of donor-host chimerism was determined 4-6 weeks or six months after injection of normal bone marrow cells into normal syngeneic or coisogeneic recipient mice. Donor-recipient pairs had chromosome markers that provided easy identification of metaphase cells. The percentage of donor cells in marrow or spleen ranged from 0 to 16% and this percentage was independent of the age of recipient or attempts to stimulate hematopoiesis in donor and/or host mice. In adult C57BL/6 mice there was a roughly linear dose-response relationship between cell dose and percentage of chimerism. There was no apparent dose-response relationship for AKR mice. The percentage of donor cells in the spleen was correlated to that seen in the marrow of recipients. Neonatal mice given the same intraperitoneal marrow cell dose as weanlings, but a larger number of cells relative to their own marrow mass, did not show a larger percentage of chimerism than weanlings. Similarly, weanlings given the same intravenous dose as adults showed no greater degree of chimerism than adults. Temporary anemia, induced by bleeding donors prior to cell collection, or more chronic hemopoietic stimulus (produced by injecting recipients with phenylhydrazine prior to cell injection with subsequent bleeding at intervals) did not result in an increased percentage of chimerism. These results indicate that there are "empty" sites in bone marrow of normal mice in which injected hematopoietic stem cells can lodge and grow.     

Extensive proliferation of subsequently injected marrow cells in parental-to-F1 hematopoietic chimeras that restored normal stem cell concentration after initial transplantation

We investigated the fate of donor stem cells that were injected into hosts with a normal concentration of spleen colony-forming unit (CFU-S). Radiation chimeras were used as hosts. When CFU-S concentration in the marrow and spleen recovered to preirradiation levels after the initial bone marrow transplantation, the subsequent transplantation was done without reirradiation. Giant granules of beige C57B1/6 (bg) mice were used as a marker and proliferation and differentiation of the stem cells of the subsequent donor origin were evaluated by measuring the proportion of neutrophils with giant granules. No beige-type neutrophils were detectable at week 24 after transplantation of 5 X 10(7) marrow cells from bg mice to intact (WB X C57B1/6)F1 (F1) mice, which were used as control recipients. In contrast, transplantation of 5 X 10(7) marrow cells to radiation chimeras resulted in the appearance of neutrophils of second-donor origin. The proportion of beige-type neutrophils was 12% at week 24 after transplantation of bg marrow cells to F1-to-F1 (syngenic) or C57B1/6-+/+-to-bg (B6-to-bg) (congenic) chimeras; the proportion of beige-type neutrophils was 43% when bg marrow cells were transplanted to B6-to-F1 semiallogenic chimeras; the proportion of normal-type neutrophils was 82% when F1 marrow cells were injected to bg-to-F1 semiallogenic chimeras. Thus, the interaction of the host hematopoietic microenvironment with the stem cells of the initial donor as well as with the stem cells of the second donor seems to influence the proliferation and differentiation of the latter stem cells.     

Organ-selective homing defines engraftment kinetics of murine hematopoietic stem cells and is compromised by Ex vivo expansion

Hematopoietic reconstitution of ablated recipients requires that intravenously (IV) transplanted stem and progenitor cells "home" to organs that support their proliferation and differentiation. To examine the possible relationship between homing properties and subsequent engraftment potential, murine bone marrow (BM) cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. PKH26(+) cells homing to marrow or spleen were then isolated by fluorescence-activated cell sorting and assayed for in vitro colony-forming cells (CFCs). Progenitors accumulated rapidly in the spleen, but declined to only 6% of input numbers after 24 hours. Although egress from this organ was accompanied by a simultaneous accumulation of CFCs in the BM (plateauing at 6% to 8% of input after 3 hours), spleen cells remained enriched in donor CFCs compared with marrow during this time. To determine whether this differential homing of clonogenic cells to the marrow and spleen influenced their contribution to short-term or long-term hematopoiesis in vivo, PKH26(+) cells were sorted from each organ 3 hours after transplantation and injected into lethally irradiated Ly-5 congenic mice. Cells that had homed initially to the spleen regenerated circulating leukocytes (20% of normal counts) approximately 2 weeks faster than cells that had homed to the marrow, or PKH26-labeled cells that had not been selected by a prior homing step. Both primary (17 weeks) and secondary (10 weeks) recipients of "spleen-homed" cells also contained approximately 50% higher numbers of CFCs per femur than recipients of "BM-homed" cells. To examine whether progenitor homing was altered upon ex vivo expansion, highly enriched Sca-1(+)c-kit+Lin- cells were cultured for 9 days in serum-free medium containing interleukin (IL)-6, IL-11, granulocyte colony-stimulating factor, stem cell factor, flk-2/flt3 ligand, and thrombopoietin. Expanded cells were then stained with PKH26 and assayed as above. Strikingly, CFCs generated in vitro exhibited a 10-fold reduction in homing capacity compared with fresh progenitors. These studies demonstrate that clonogenic cells with differential homing properties contribute variably to early and late hematopoiesis in vivo. The dramatic decline in the homing capacity of progenitors generated in vitro underscores critical qualitative changes that may compromise their biologic function and potential clinical utility, despite their efficient numerical expansion.     

The hematopoietic stem cells of alpha-thalassemic mice

The alpha-thalassemic mouse has a hereditary microcytic anemia, almost certainly has a shortened RBC life span, and is a potential candidate for cell replacement therapy. In a routine study of bone marrow repopulating capacity using hemoglobin as a cell marker, normal donor marrow cells, but not alpha-thalassemic donor marrow cells, completely replaced the host cells. Further analysis showed that at least 30 times more alpha-thalassemic cells were required to outcompete normal donor cells injected simultaneously. The results were more extreme then expected and suggested a defect in a stem cell population as well as in the RBCs. Evidence that the multipotent and erythroid-committed stem cells in alpha-thalassemic mice are not decreased was shown by CFU-S and CFU-E assays. The combined results indicate that the deletion expresses itself most conspicuously in the RBC population. Tests were also performed to analyze repopulation kinetics in the Hbath-J/+ mice. In unirradiated alpha-thalassemic hosts, the hemoglobin from a normal donor persisted but did not replace the host hemoglobin. Sublethally irradiated alpha-thalassemic hosts, on the other hand, were easily repopulated with normal cells. We conclude that the alpha-thalassemic mouse is a good model for cell replacement therapy.     

Effects of the motheaten gene on murine B-cell production

The rapidly fatal autoimmune disease in the mutant mouse known as motheaten is caused by an autosomal recessive gene and is characterized by hypergammaglobulinemia and autoantibody production, among other defects. The cellular kinetics of B-cell maturation were investigated in three-week-old motheaten mice and their normal littermates to determine whether any abnormality in cell production of the B lineage could be correlated with B-cell hyperactivity. The production rates and renewal times of newly produced bone marrow, splenic small B-lymphocytes, and splenic plasma cells were examined by in vivo tritiated-thymidine administration using a pulse-chase protocol and radioautography of immunofluorescence-stained cells. Because small B-lymphocytes in both organs were produced at comparable rates in the mutant mice and in their normal littermates, primary B-cell production was unaffected in the mutant mice. In contrast, splenic plasma cells were produced 10-30 times faster in motheaten mice than in normal mice. The enhanced rate of plasma cell production in motheaten mice could be correlated with a concurrent increased loss of labeled large B-lymphocytes, presumably rapidly dividing activated B cells. Thus, the excessive antibody production in motheaten mice may be reflected by the increased plasma cell production.     

The detection of normal hidden stem cells during the development of leukemia: assays with PGK isozyme

We evaluated the changes in the number of normal spleen colony-forming units (CFU-S) in the spleen and the bone marrow of C3H/He mice during the development of leukemia following the injection of the murine leukemia cell line, MK-8057, MK-8057 cells, originating in C3H/He PGK-1b mice, were injected into syngeneic C3H/He PGK-1a mice so that phosphoglycerate kinase (PGK) isozymes could be used to distinguish leukemic spleen colonies from normal colonies when cells from the spleen or bone marrow of the recipients were reinjected into lethally irradiated mice. Leukemic cells showed a logarithmic increase in the recipient mice and had replaced the bone marrow and the spleen completely by days 6-8; the mice started to die of leukemia after day 11. However, colonies examined from normal stem cells still comprised 60% of the total number of spleen colonies on day 6, 45% on day 8, and 20% on day 10. Furthermore, when the numbers of normal CFU-S were calculated as numbers per spleen, we found that they increased exponentially to a level 100 times higher than the normal level 10 days after injection of MK-8057 cells.     

Smad5 is dispensable for adult murine hematopoiesis

Smad5 is known to transduce intracellular signals from bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-beta (TGF-beta) superfamily and are involved in the regulation of hematopoiesis. Recent findings suggest that BMP4 stimulates proliferation of human primitive hematopoietic progenitors in vitro, while early progenitors from mice deficient in Smad5 display increased self-renewal capacity in murine embryonic hematopoiesis. Here, we evaluate the role of Smad5 in the regulation of hematopoietic stem cell (HSC) fate decisions in adult mice by using an inducible MxCre-mediated conditional knockout model. Surprisingly, analysis of induced animals revealed unperturbed cell numbers and lineage distribution in peripheral blood (PB), bone marrow (BM), and the spleen. Furthermore, phenotypic characterization of the stem cell compartment revealed normal numbers of primitive lin(-)Sca-1(+)c-Kit(+) (LSK) cells in Smad5(-)(/)(-) BM. When transplanted in a competitive fashion into lethally irradiated primary and secondary recipients, Smad5-deficient BM cells competed normally with wild-type (wt) cells, were able to provide long-term reconstitution for the hosts, and displayed normal lineage distribution. Taken together, Smad5-deficient HSCs from adult mice show unaltered differentiation, proliferation, and repopulating capacity. Therefore, in contrast to its role in embryonic hematopoiesis, Smad5 is dispensable for hematopoiesis in the adult mouse.     

Self-renewal and differentiation capacity of bone marrow and fetal liver stem cells

S ummary . An operational definition of the pluripotent stem cell (CFC-S) requires that it have both the capacity for self-renewal and the potential for differentiation into more than one class of formed blood elements. Because the CFC-S compartment is heterogeneous, younger stem cells would be expected to be less committed to differentiation and have a higher rate of self-renewal; whereas, older stem cells would be more committed to differentiation and have a lower rate of self-renewal. In this study, the self-renewal capacity versus the differentiation potential of adult bone marrow and fetal liver stem cells were compared. The self-renewal potential was estimated by determining the number of CFC-S which develop during growth in the spleen or femur of primary recipients. The differentiation potential was estimated by determining the total number of nucleated cells or committed progenitor cells (GM-CFU-C and BFU-E) which develop during growth in the spleen or femur of primary recipients. In order to circumvent possible differences in self-renewal or differentiation pressures due to the presence of differing numbers of CFC-S, an equivalent number of bone marrow and fetal liver CFC-S were allowed to seed the spleen and femur. While both adult bone marrow and fetal liver stem cells showed an extensive capacity for self-renewal, fetal liver CFC-S displayed a greater potential for self-renewal in both the spleen and femur and at all growth intervals measured when compared to adult bone marrow CFC-S. In contrast, no differences were seen in the number of nucleated cells or committed stem cells found per CFC-S when comparing adult bone marrow and fetal liver stem cells.     

Quantitative genetic variation in the hematopoietic stem cell and progenitor cell compartment and in lifespan are closely linked at multiple loci in BXD recombinant inbred mice

The number of bone marrow hematopoietic stem and progenitor cells as defined by the lineage(-), Sca1(++), c-kit(+) (LSK) phenotype and their proliferative capacity in vitro are subject to quantitative genetic variation, and several quantitative trait loci (QTL) have been identified in young mice. Because some traits affecting hematopoiesis also change with age in a mouse strain-dependent fashion, we performed quantitative trait analysis in aged BXD recombinant inbred (RI) mice for the number and frequency of LSK cells, and for their proliferative capacity in vitro. Several novel QTL were identified. The number and frequency of LSK cells in old mice correlated inversely with lifespan. Furthermore, 4 of 7 lifespan QTL overlap with QTL contributing to the number, frequency, or proliferative capacity of LSK cells in young or old mice. Taken together, these data establish a close genetic, and perhaps functional, link between genetic variation in lifespan and characteristics of stem and progenitor cells.     

Thrombopoietin signaling is required for in vivo expansion of IL-11--responsive hematopoietic progenitor cells in the steady state

OBJECTIVE: mpl(-/-) mice have a profound defect in platelets and megakaryocytes and a defect in hematopoietic progenitor cells and stem cells. However, no specific subset of the progenitor/stem cell compartment has been shown to be particularly affected by this deficiency in mpl(-/-) mice. In this article, we identified a specific subset of bone marrow progenitor/stem cells that was altered in mpl(-/-) mice. MATERIALS AND METHODS: In vitro and in vivo hematopoietic assays were utilized to examine the response to interleukin-11 in mice lacking the receptor for thrombopoietin (TPO) (mpl(-/-) mice). RESULTS: The interleukin (IL)-11-responsive subset of progenitor cells was not detected in clonal cultures of bone marrow cells from mpl(-/-) mice. However, mpl(-/-) mice responded to IL-11 in vivo as evidenced by a rise in platelet count and an increase in spleen weight. Experiments were performed to address this paradox: administration of 5-fluorouracil with consequent "expansion" of early hematopoietic cells resulted in the appearance of IL-11-responsive cells in mpl(-/-) mice when assayed in in vitro cultures. CONCLUSIONS: Thus, although mpl(-/-) mice have the capacity to produce IL-11-responsive progenitor cells, under steady state conditions their expansion is dependent on TPO. This is the first evidence that a specific subset of bone marrow progenitor/stem cells is altered in mpl(-/-) mice.