Analysis of the hematopoietic effects of new dominant spotting (W) mutations of the mouse. II. Effects on mast cell development

In addition to their anemia, sterility and lack of coat pigment (1,2), W/Wv mice are mast cell deficient (3,4). Our analysis of three recently described W alleles (5) confirms reports (3,6) that (a) W mutations alter skin mast cell number in parallel with their influence on red cell number (but not with pigmentation), (b) that mast cells arise from hematopoietic tissue (7) and (c) that injections of normal bone marrow cells, which cure the anemias of W/Wv recipients, also alleviate the deficiency of skin mast cells in these mice. Transplants of bone marrow cells from mice homozygous for two new anemia-causing W alleles, W39 and W41, fail to cure the anemias of W/Wv recipients (companion paper) or increase the number of mast cells in their skin. Marrow cell implants from non-anemic W44/W44 mice cure the anemia, but do not change the number of mast cells in the skin of W/Wv recipients. The fact that the bone marrows of all three new homozygotes have fewer than normal numbers of CFUs hematopoietic stem cells (see companion paper) and have reduced mast cell-regenerating capacities, supports Kitamura's contention (8) that mast cell precursors may be closely related to or identical with the CFUs.     

Prevention of genetic anemias in mice by microinjection of normal hematopoietic stem cells into the fetal placenta

Mice homozygous for mutant genes at the W locus have a marked macrocytic anemia that is fatal in some genotypes. The defect is believed to originate in the developmentally pluripotent hematopoietic stem cell population. Anemia is first grossly manifest on day 13 of gestation, when the liver is the chief hematopoietic organ. The known paucity of blood-forming foci in livers of homozygotes and the limited formation of their erythrocytes suggested that such fetuses-unlike normal ones-might have conditions favorable for in utero seeding of genetically normal hematopoietic tissue. If this were accomplished before day 13, the anemia might essentially be prevented, or at least substantially mitigated, and normalcy soon achieved by cell selection. This proved to be the case. Allogeneic normal fetal liver cells were microinjected into the blood vessels of the fetal placenta on day 11 of gestation. Of eight mutant homozygotes born from segregating matings, six (four W/W, two W(v)/W(v)) were successfully populated with donor cells. Strain-specific hemoglobin markers demonstrated replacement of the erythroid lineage with the normal type, the rate of substitution being more rapid in the W/W (ordinarily more anemic) recipients. Strain-specific isozyme differences revealed that white blood cells were also replaced. Thus, the initial selective pressure, hence the W-mutant phenotypic lesion, must have occurred at the pluripotent stem cell stage. The animals remained immunologically tolerant of the donor cells and no graft-versus-host reaction occurred. The early introduction of hematopoietic cells differing genetically from all the other tissues of the animal provides possibilities for tracing normal hematopoietic lineages in vivo, for analyzing cell and tissue interactions, such as those between lymphocytes and thymus, and for clarifying the etiology of other blood or immune insufficiencies or malignancies.     

Hematopoietic Cells From alpha -Spectrin-Deficient Mice Are Sufficient to Induce Thrombotic Events in Hematopoietically Ablated Recipients

Thrombotic events are life-threatening complications of humanhemolytic anemias such as paroxysmal nocturnal hemoglobinuria, sicklecell disease, and thalassemia. It is not clear whether these events aresolely influenced by aberrant hematopoietic cells or also involveaberrant nonhematopoietic cells. Spherocytosis mutant(Spna1^sph/Spna1^sph; for simplicityreferred to as sph/sph) mice develop a severe hemolytic anemiapostnatally due to deficiencies in -spectrin in erythroid and otheras yet incompletely defined nonerythroid tissues. Thrombotic lesionsoccur in all adult sph/sph mice, thus providing ahematopoietically stressed model in which to assess putative causes ofthrombus formation. To determine whether hematopoietic cells fromsph/sph mice are sufficient to initiate thrombi, bone marrowfrom sph/sph or +/+ mice was transplanted into mice with nohemolytic anemia. One set of recipients was lethally irradiated; theother set was genetically stem cell deficient. All mice implanted withsph/sph marrow, but not +/+ marrow, developed severe anemia and histopathology typical of sph/sph mice. Histologicalanalyses of marrow recipients showed that thrombi were present in therecipients of sph/sph marrow, but not +/+ marrow. Theresults indicate that the -spectrin-deficient hematopoietic cellsof sph/sph mice are the primary causative agents of thethrombotic events.     

Latent deficiency of the hematopoietic microenvironment of aged mice as revealed in W/Wv mice given +/+ cells

The macrocytic anemia of W/Wv mice can be cured by injection of +/+ bone marrow cells (BMC) from WBB6F1 mice. However, it has been observed that some W/Wv recipients appear to "lose" their cure with time, an effect that does not appear to be related to the age of the BMC donor. The present study was undertaken to determine the effect of recipient age on W/Wv responses to BMC injection. The effect of aging on erythroid parameters was similar in untreated W/Wv mice and +/+ controls. In both genotypes, hematocrit (HCT) and red blood cell count (RBC) decreased, and the modal red blood cell size (peak) increased between 13 and 150 weeks of age. As anticipated, mean HCT and RBC values were lower and peak values higher in W/Wv mice compared to +/+ controls at every age. However, the rate of decrease in HCT and RBC with age was the same for both genotypes, suggesting that the age effect and W gene effect were independent. Peak values increased slightly more with age for W/Wv than for +/+ controls. When female W/Wv mice in three age groups (23.5, 70, and 91.5 weeks old) were injected with 5 x 10(5) BMC from 20-week-old +/+ female donors and HCT, RBC, and peak were determined monthly, improvement was seen in most W/Wv recipients. However, in the older mice this improvement was slower and often was not sustained; 100% of the youngest recipients, 80% of the middle-aged, and only 30% of the older groups were cured after 3 months. Taken together, these data suggest a latent deficiency of the aging hematopoietic microenvironment that is revealed in W/Wv mice by the stress of continuing erythroid demand on the limited number of normal donor BMC.     

In vivo expansion of purified hematopoietic stem cells transplanted in nonablated W/Wv mice

We have evaluated the in vivo amplification potential of purified murine hematopoietic stem cells, identified as Wheat Germ Agglutinin+ (WGA+), 15-1.1(-) , Rhodamine 123 Dull (Rho-dull) cells, by serial transplantation into stem cell defective nonmyeloablated W/Wv mice. C57BL Rho-dull cells (250/ 500 cells/mouse) permanently engrafted nonablated W/Wv mice as defined by the presence of > 95% red and > 20% white donor-derived circulating cells for at least 1.5 years following transplantation. At this time, approximately 61% of Rho-dull cells and all the Rho-bright progenitor and colony forming cells of the engrafted mice were found to be donor-derived by c-Kit genotyping and by their response to stem cell factor (SCF). Retransplantation of 250-1000 Rho-dull cells from primary into secondary W/Wv recipients generated C57BL hematopoiesis in 40%-64% of animals revealing the presence of donor derived hematopoietic stem cells (HSC) in the bone marrow of the primary recipients. One and half years after transplantation, the bone marrow of the secondary engrafted animals contained C57BL Rho-dull cells approximately = 51% by genotype), which were capable of reconstituting tertiary W/Wv recipients. In this respect, 25% of tertiary mice expressed C57BL hematopoiesis when transplanted with 250-1000 Rhodull cells purified from secondary W/Wv recipients. On the basis of the number of Rho-dull cells purified from a single mouse, we calculate that approximately 7.3x10(4) Rho-dull cells, which are genotypically and functionally defined as C57BL long-term repopulating stem cells, were generated in the marrow of reconstituted primary W/Wv recipients transplanted 1.5 years earlier with 250-500 C57BL Rho-dull cells. We conclude that murine HSC have extensive amplification capacity in nonmyeloablated animals.     

Engraftment of bone marrow transplants in W anemic mice measured by electronic determination of the red blood cell size profile.

Defective stem cells of WBB6F1-W/Wv mice produce macrocytic red blood cells (RBCs); stem cells of WBB6F1-+/+ mice produce normocytic RBCs. Utilization of the Coulter counter channelyzer permitted good dissociation between the size distribution of populations of +/+ and W/Wv RBCs. Peaks (mean cell volumes) for +/+ and W/Wv RBCs have been determined to be between the 30th and 40th channel and 50th and 60th channel, respectively. Variability of profiles for individual mice of both genotypes did not exceed the variability of separate determinations of the same cell suspension from a single mouse. Admixture (approximately 15%) of either type of erythrocytes could be quantitatively detected by this method. One week after transplant of 10(7) +/+ marrow cells into W/Wv recipients, 25% of donor type erythrocytes were detected. Eighteen days post-graft, concentration of +/- normocytes exceeded the concentration of macrocytes in the W/Wv recipients' circulation. Approximately 45 days post-transplant, the proportion of macrocytes decreased below the 10% detectable level. Calculation of the daily RBC production rate during repopulation and estimation of the number of RBCs produced by a single hematopoietic colony were determined. The RBC size profile was found to be a convenient method for studying the effect of implantation of W/Wv marrow into lethally irradiated +/+ mice. This method proved suitable for repetitive determination of the size population in individual transplanted mice.     

Heterozygous kit mutants with little or no apparent anemia exhibit large defects in overall hematopoietic stem cell function

OBJECTIVE: The evolutionarily conserved Kit receptor is vital for function of hematopoietic stem cells (HSC). Kit(W-41) (W-41) and Kit(W-42) (W-42) are single residue changes in the KIT intracellular phosphotransferase domain, while Kit(W-v) (W-v) is a single residue change in the ATP binding domain. This study tests how each mutation affects HSC function. METHODS: Cells in mutant and C57BL/6J(+/+) blood and marrow were compared. Overall HSC function was measured by competitive repopulation. Functions of specific progenitor populations were tested with stage-specific competitive repopulation and standard colony-forming unit assays. RESULTS: Bone marrow cells from these Kit mutants are severely defective at reconstituting peripheral blood lineages and bone marrow of irradiated recipients, when compared to +/+ control marrow. These defects increased with time. Marrow from W-41/+ and W-v/+ functions similarly but better than marrow from W-41/W-41 and W-42/+, to repopulate the erythroid and lymphoid lineages. Long-term (LT) and short-term (ST) HSC from W-v/+, W-41/W-41, and W-42/+ are more defective at reconstituting bone marrow than LT- and ST-HSC from W-41/+ and +/+. Common myeloid progenitor (CMP) cells from W-42/+ and W-41/W-41 are more defective at producing spleen colonies than CMP from W-v/+ and W-41/+. CONCLUSION: Heterozygous Kit mutants with little or no apparent anemia exhibit surprisingly large defects in overall HSC function. Multiplying the fractional defects in LT-HSC, ST-HSC, and CMP can account for overall effects of W-v/+, but does not completely account for the defects observed with W-41/+, W-42/+, and W-41/W-41.     

Reversible expression of CD34 by murine hematopoietic stem cells.

We used a mouse transplantation model to address the recent controversy about CD34 expression by hematopoietic stem cells. Cells from Ly-5.1 C57BL/6 mice were used as donor cells and Ly-5.2 mice were the recipients. The test cells were transplanted together with compromised marrow cells of Ly-5.2 mice. First, we confirmed that the majority of the stem cells with long-term engraftment capabilities of normal adult mice are CD34(-). We then observed that, after the injection of 150 mg/kg 5-fluorouracil (5-FU), stem cells may be found in both CD34(-) and CD34(+) cell populations. These results indicated that activated stem cells express CD34. We tested this hypothesis also by using in vitro expansion with interleukin-11 and steel factor of lineage(-) c-kit(+) Sca-1(+) CD34(-) bone marrow cells of normal mice. When the cells expanded for 1 week were separated into CD34(-) and CD34(+) cell populations and tested for their engraftment capabilities, only CD34(+) cells were capable of 2 to 5 months of engraftment. Finally, we tested reversion of CD34(+) stem cells to CD34(-) state. We transplanted Ly-5.1 CD34(+) post-5-FU marrow cells into Ly-5.2 primary recipients and, after the marrow achieved steady state, tested the Ly-5.1 cells of the primary recipients for their engraftment capabilities in Ly-5.2 secondary recipients. The majority of the Ly-5.1 stem cells with long-term engraftment capability were in the CD34(-) cell fraction, indicating the reversion of CD34(+) to CD34(-) stem cells. These observations clearly demonstrated that CD34 expression reflects the activation state of hematopoietic stem cells and that this is reversible.     

Processing by the thymus is not required for cells that cure and populate W/WV recipients.

Adult marrow, fetal liver or nu/nu mouse marrow from histocompatible donors was grafted into genetically anemic W/WV recipients, and all three types of grafts cured thymectomized as well as intact W-anemic recipients. With the latter two types of graft, the genetic anemia was cured by cells that could not have been processed in a mature thymus, since the adult recipients were thymectomized before receiving the grafts, the nu/nu donors were congenitally thymusless, and the fetal donors were used at 16 days of gestation. Chromosome-marked marrow grafts were used to show that immune systems were populated to similar degrees in thymectomized and intact W/WV recipients. Therefore, the cells derived from the donor marrow graft that partially populate the immune systems of W-anemic recipients do not require thymus processing. Small numbers of liver rudiment or yolk sac cells from fetal donors less than 12 days old failed to cure W/WV recipients, even when mixed with adult thymus cells. Therefore, the lack of adequately developed thymic helper cells appears not to be the reason why early fetal hemopoietic stem cells fail to cure W/WV recipients.     

Telomere length changes in patients undergoing hematopoietic stem cell transplantation.

Telomere length indicates the replicative history of cells, serving as a molecular measure of the replicative potential remaining in cells. To investigate telomere length changes in hematopoietic stem cells, patients undergoing hematopoietic stem cell transplantation (HSCT) were evaluated. Fifteen patients after allogeneic bone marrow transplantation (allo-BMT group), seven patients after autologous peripheral blood stem cell transplantation (auto-PBSCT group), and 39 healthy controls were studied. Telomere length was measured in peripheral mononuclear cells by Southern blot hybridization. There was no significant difference between the allo-BMT and the auto-PBSCT groups. In the allo-BMT group, the mean telomere length of recipients was 2.01 kb shorter than that of their donors (P = 0. 008), and was 1.59 kb shorter than that of age-matched putative normal controls (P = 0.002). Telomere shortening in the allo-BMT group was equivalent to 41.4 years of aging in the donors, and to 52. 4 years of aging in the normal controls. The mean telomere length in the auto-PBSCT group was 2.36 kb shorter than that of the age-matched putative controls (P = 0.043), which was equivalent to 61.5 years of aging in normal controls. The extent of telomere shortening in the allo-BMT group showed a trend to negative correlation with the number of mononuclear cells infused. These findings suggest that hematopoietic stem cells after HSCT lose telomere length and these shortened telomeres may result in a higher incidence of clonal disorders later in life.     

Effects of transplantation and age on immunohemopoietic cell growth in the splenic microenvironment

Intact spleens from young adult and aged mice were transplanted into young recipients to compare effects of age and effects of spleen transplantation on hemopoietic and immune functions. Hemopoietic functions of histocompatible spleen transplants were assessed by partial cures of genetically anemic WBB6F1-Sl/Sld recipients, and immune functions were measured as numbers of anti-SRBC PFC(sheep red blood cell plaque-forming cells) and responses to the mitogen PHA (phytohemagglutinin). Spleens from WCB6F1 and WBB6F1 donors at least 28 months old partially corrected anemias in 10 of 28 Sl/Sld recipients, whereas spleens from 5- to 10-month-old donors performed significantly better, partially correcting anemias in 22 of 31 Sl/Sld recipients. B6D2F1 spleens were transplanted from either old or young donors in B6D2F1 recipients to test their ability to support immune-responsive cells. These spleen grafts were much smaller than recipient spleens and contained few anti-SRBC PFC. In contrast WCB6F1-+/+ spleens transplanted in Sl/Sld recipients were much larger, weighing more than the intact spleens of the recipients. Nevertheless when these spleens were from young donors, they contained only about 10% as many anti-SRBC PFC and PHA-responsive cells as did recipient spleens, whereas old donor spleens contained even fewer. Use of splenectomized Sl/Sld recipients did not alter these results. Apparently the effect of transplantation was much more important than age in reducing the spleens' abilities to support immune-responsive cells.     

Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice.

MRL/MP-+/+ (MRL/+) mice develop pancreatitis and sialoadenitis after they reach 7 months of age. Conventional bone marrow transplantation has been found to be ineffective in the treatment of these forms of apparent autoimmune disease. Old MRL/+ mice show a dramatic thymic involution with age. Hematolymphoid reconstitution is incomplete when fetal liver cells (as a source of hemopoietic stem cells) plus fetal bone (FB; which is used to recruit stromal cells) are transplanted from immunologically normal C57BL/6 donor mice to MRL/+ female recipients. Embryonic thymus from allogeneic C57BL/6 donors was therefore engrafted along with either bone marrow or fetal hematopoietic cells (FHCs) plus fragments of adult or fetal bone. More than seventy percent of old MRL/+ mice (> 7 months) that had been given a fetal thymus (FT) transplant plus either bone marrow or FHCs and also bone fragments survived more than 100 days after treatment. The mice that received FHCs, FB, plus FT from allogeneic donors developed normal T cell and B cell functions. Serum amylase levels decreased in these mice whereas they increased in the mice that received FHCs and FB but not FT. The pancreatitis and sialoadenitis already present at the time of transplantations were fully corrected according to histological analysis by transplants of allogeneic FHCs, FB and FT in the MRL/+ mice. These findings are taken as an experimental indication that perhaps stem cell transplants along with FT grafts might represent a useful strategy for treatment of autoimmune diseases in aged humans.     

Differential role for very late antigen-5 in mobilization and homing of hematopoietic stem cells

The role of very late antigen-5 (VLA-5) in homing and mobilization of hematopoietic stem cells from normal bone marrow (NBM) and bone marrow (MBM) and peripheral blood (MPB) from mobilized mice was investigated. We found a decreased number of VLA-5-expressing cells in the lineage-negative fraction of MPB. However, virtually all stem/progenitor cells were present in the VLA-5(+) fraction and hence mobilization of hematopoietic stem cell subsets does not coincide with a downregulation of VLA-5. Stem/progenitor cells from MPB and MBM demonstrated enhanced stromal-derived factor-alpha-induced migration. This enhanced migration correlates with an improved hematopoietic reconstitution potential, with the migrated MPB cells showing the fastest reconstitution. Interestingly, homing of MPB, MBM and NBM stem/progenitor cells in bone marrow and spleen did not differ and is therefore not responsible for the differences in hematopoietic reconstitution. The observed increase in VLA-5(+) cells in the recipients after transplantation can most probably be attributed to selective homing of VLA-5(+) cells instead of an upregulation of VLA-5. Treatment with an antibody to VLA-5 partially inhibited bone marrow homing of progenitor cells, whereas homing in the spleen was hardly affected. These data indicate a differential role for VLA-5 in the movement of stem cells from and toward bone marrow.     

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.     

Adenovirus infections in hematopoietic stem cell transplant recipients.

We report a 12% incidence of adenovirus infections among 532 recipients of hematopoietic stem cell transplant (HSCT) from January 1986 through March 1997. The median time from day of stem cell infusion to first positive culture was 41 days. Recipients of allogeneic stem cells, as opposed to autologous stem cell recipients, were more likely to have a culture positive for adenovirus (16% vs. 3%; P<.0001). Pediatric patients were also more likely than adults to have a positive culture (23% vs. 9%; P<.0001). Among stem cell recipients with partially matched related donors, pediatric recipients appear to be at significantly greater risk for infection than adult recipients (P<.001). Positive cultures were associated with evidence of invasion in 64% of cases (41 of 64). A multiple logistic regression analysis showed that isolating adenovirus from more than 1 site correlated with greater risk for invasive infections (P=.002). Invasive infections were associated with poorer chance of survival.     

Effect of rabbit antimouse brain serum on marrow cells capable of curing W/Wv mice

The W/Wv mouse has a recessively inherited defect in hematopoietic stem cells (HSC) but can be cured of its hematopoietic abnormalities by infusion of marrow from a co-isogeneic, +/+ mouse. The "curative" cell for the W/Wv is thought to be a subcompartment of the HSC that is capable of forming hematopoietic spleen colonies (CFU-S) in irradiated mice. The curative HSC must have a very high proliferative potential and it is known that HSC with variable degrees of proliferative potential are found within the CFU-S compartment. Rabbit antimouse brain serum (RAMBS) was used to treat +/+ marrow and its effect upon CFU-S and upon curative cells was compared with the effect of normal rabbit serum (NRS) or of sham treatment. CFU-S were reduced to 70%-79% of control by NRS and to 8%-9% by RAMBS. Curative cells for the W/Wv were not detectably reduced by NRS; they were reduced by RAMBS, but to only approximately 20%-30% of control. Thus, it appeared to a certain degree that RAMBS spared HSC with a high proliferative potential when compared with its effect on the entire CFU-S compartment.     

Bystander destruction of hematopoietic progenitor and stem cells in a mouse model of infusion-induced bone marrow failure

Infusion of parental lymph node (LN) cells into sublethally irradiated hybrid F1 recipients created a murine model for bone marrow (BM) failure. Affected animals developed fatal pancytopenia within 2 to 3 weeks, accompanied by BM oligoclonal T-cell infiltration and severe marrow hypoplasia indicated by approximately 10-fold declines in total BM cellularity, 15-fold declines in BM Lin(-)Sca1(+)c-Kit(+) cells, 100-fold declines in spleen colony-forming units, and 100-fold declines in hematopoietic progenitor and stem cells as estimated by irradiation protection in vivo. LN cells of both H2(b/b) and H2(d/d) haplotypes were effectors. Serum interferon-gamma (IFN-gamma) concentration increased 2- to 3-fold. Marrow cells were severely apoptotic, with high proportions of Fas(+) and annexin V(+) cells. Cotransplantation of 5 x 10(5) BM cells from clinically affected donors and 10(6) BM cells from H2 identical healthy mice could not rescue lethally irradiated recipients. Recipients had significantly lower cellularity in peripheral blood and BM, and cell mixtures failed to produce a stromal feeder layer to support marrow cell growth in vitro. Pathogenic T cells from donors after BM failure appeared capable of destroying hematopoietic progenitor, stem, and stromal cells from fully compatible healthy donors as "innocent bystanders." This effect can be partially abrogated by anti-IFN-gamma antibody.     

Transplantation of wheat germ agglutinin-positive hematopoietic cells to prevent or induce systemic autoimmune disease.

Hematopoietic stem cell defects are thought to be involved in the etiopathogenesis of systemic autoimmune disease. Positively selected, stem cell-enriched populations of wheat germ agglutinin-positive (WGA+) low-density bone marrow and fetal liver cells from normal and autoimmune-prone mice were used to determine whether reciprocal transplantation of stem cells between normal and autoimmune-prone mice inhibits or causes development of autoimmune disease. NZB recipients of DBA/2 stem cell populations analyzed greater than 100 days after bone marrow or fetal liver cell transplantation showed decreased levels of anti-DNA antibodies and decreased glomerular lesions when compared with nontreated NZB mice or NZB recipients of NZB stem cell preparations. Female DBA/2 recipients of WGA+ NZB bone marrow cell or fetal liver cell transplants exhibited elevated serum autoantibody levels and developed glomerular lesions characteristic of NZB mice when analyzed greater than 100 days after transplantation. These pathological disturbances were not observed in DBA/2 recipients of DBA/2 stem cell preparations. The data indicate that WGA+ stem cells from autoimmune-prone NZB mice contain the genetic defects responsible for the development of systemic autoimmune disease.     

Normal Production of Erythrocytes by Mouse Marrow Continuous for 73 Months

Marrow cell transplants from old and young control donors were carried in genetically anemic W/W(v) recipients whose anemias were cured by successful transplants. After maximum of 36 months and four serial transplants, marrow cell lines from both old and younger control donors continued to produce erythrocytes normally. The oldest marrow cell lines had produced erythrocytes normally for 73 months.NORMAL ERYTHROCYTE PRODUCTION WAS DEMONSTRATED BY: (1) cure of the anemia in W/W(v) recipients, (2) normal rather than delayed recovery rate of cured recipients after severe bleeding, and (3) normal rather than ineffective response of cured recipients to erythropoietin. Hemoglobin patterns, tested in cured W/W(v) recipients after the first transplantation, showed that at least 90% of the circulating erythrocytes were of the donor type even in donor lines that had produced erythrocytes continuously for 45 months and were recovering from severe bleeding. Concentrations of cells capable of forming macroscopic spleen colonies were more than two orders of magnitude higher in W/W(v) mice cured by old or younger marrow than in uncured W/W(v) mice. Nevertheless, colony-forming unit concentrations declined slowly with successive transplants, and the decline seemed more pronounced at the fourth transplant in old than in younger cell lines.The hypothesis is suggested that senescence is caused by declines in function of only a few vital cell types. The system for comparing old and younger marrow cell lines offers a model for experiments to test this hypothesis and to identify the cell types whose decline causes aging.     

Progenipoietin-1: a multifunctional agonist of the granulocyte colony-stimulating factor receptor and fetal liver tyrosine kinase-3 is a potent mobilizer of hematopoietic stem cells

OBJECTIVE: Progenipoietin-1 is an agonist of both the granulocyte colony-stimulating factor and fetal liver tyrosine kinase-3 receptors capable of inducing the proliferation of multiple hematopoietic cell lineages. The potential of progenipoietin-1 to mobilize transplantable hematopoietic stem cells into the peripheral blood was evaluated. METHODS: Cohorts of donor mice were treated with either progenipoietin-1, fetal liver tyrosine kinase-3 ligand, granulocyte colony-stimulating factor, or a vehicle control. Hematopoietic progenitor/stem-cell activity in donor blood was assayed by radioprotection, multilineage reconstitution, secondary transplantation, and competitive repopulation. RESULTS: Only 1 microL of peripheral blood from progenipoietin-1-treated donors was required to protect 80% of lethally irradiated mice, while in contrast 1 microL of peripheral blood from granulocyte colony-stimulating factor-treated donors failed to protect any recipients. The radioprotected recipients of progenipoietin-1-treated donor cells showed donor-derived (Ly5.2) multilineage hematopoietic reconstitution for up to 6 months. Serial transplantation studies using bone marrow from radioprotected, chimeric recipients demonstrated long-term donor-derived hematopoiesis, indicating the successful transplantation of multipotent hematopoietic stem cells. The engraftment potential of progenipoietin-1 donor-derived cells was directly compared with donors treated with granulocyte colony-stimulating factor or fetal liver tyrosine kinase-3 ligand alone or in combination. Both spleen colony-forming activity and competitive repopulating activity was highest in the blood from progenipoietin-1-treated donors. CONCLUSIONS: These studies demonstrate that progenipoietin-1 is a potent mobilizer of transplantable hematopoietic stem cells and indicate that this dual-receptor agonist has greater biologic activity than its constituent molecules.