Recent progress in identifying genes regulating hematopoietic stem cell function and fate.

Significant advances in the use of genetic and molecular biology strategies have recently begun to identify genes that have a major impact on the determination, commitment and developmental potential of hematopoietic stem cells. Using a variety of experimental strategies, genes such as SCL, GATA-2, HoxB4, Flk-2, c-mpl, dlk, and others have been implicated as important regulators of stem cell growth. In addition, genetic mapping has identified several loci that correlate strongly with stem cell numbers and proliferation.

Studies of hematopoietic stem cells spared by 5-fluorouracil.

Mouse marrow cells were exposed to 5-fluorouracil (FU) either in vivo or in vitro and the effects on the hematopoietic stem cell compartment were studied. The drug was highly toxic to bone marrow cells including the spleen colony-forming unit (CFU-S) population. The small population of stem cells surviving FU, however, caused a different pattern of spleen colony growth when injected into lethally irradiated mice. Whereas numbers of spleen colonies caused by normal marrow cells remained constant during an 8-14 d period after transplantation, spleen colonies derived from FU-treated marrow cells increased by as much as 100-fold during this time. This effect on stem cells was dose dependent both in vitro and in vivo. When FU was given in vivo, the day 14/day 8 ratio of colonies was greatest 1 d after injection and, over the next 7 d, returned to a near-normal value, that is, unity. A number of studies have shown that the stem cell compartment is heterogeneous with respect to self-replicative capacity and developmental potential. An age structure for the stem cell compartment has been proposed wherein cells with a short mitotic history are more likely to self-replicate than they are to differentiate; hence they are more primitive. 'Older' stem cells with a longer mitotic history are, according to the hypothesis, more likely to differentiate. 5-fluorouracil may be toxic to the older stem cells and selectively spare the more primitive subpopulation. Although the surviving cells may not themselves be able to form spleen colonies, they may give rise to an older cohort of cells more likely to differentiate and form spleen colonies. It is the requisite developmental maturation within the stem cell compartment that may be responsible for the delay in appearance of spleen colonies derived from FU-treated marrow. Our results support this explanation and identify the locus of at least part of this activity as the bone marrow. We found that the FU-treated marrow did not cause an increase in spleen colony numbers between 8 and 14 d in hosts with a long-standing marrow aplasia, due to the incorporation of 89Sr into bone. I propose that the delayed spleen colony appearance in normal hosts is the result of developmental maturation of the primitive stem cell compartment that survives FU and is responsible for spleen colonies arising around day 14. This maturation, at least initially, occurs in the marrow and leads to the replenishment of the more differentiated CFU-S subsets ablated by FU, which are normally responsible for spleen colonies appearing earlier after transplantation.

Intrinsic and extrinsic control of hemopoietic stem cell numbers: mapping of a stem cell gene.

We evaluated in vivo interactions between extrinsic (growth factor induced) and intrinsic (genetically determined) effectors of mouse primitive hemopoietic stem cell proliferation and numbers. Accordingly, stem cell frequency and cell cycle kinetics were assessed in eight strains of inbred mice using the cobblestone area-forming cell (CAFC) assay. A strong inverse correlation was observed between mouse lifespan and the number of autonomously cycling progenitors (CAFC day 7) in the femur. The population size of primitive stem cells (CAFC day 35) varied widely (up to sevenfold) among strains, unlike total CAFC day 7 numbers (cycling and quiescent), which were similar. Administration of the early acting cytokine flt-3 ligand to these strains resulted in activation of quiescent primitive stem cells exclusively in strains with high endogenous stem cell numbers (DBA and AKR), but was unrelated to strain-specific progenitor cell cycling. To map loci affecting stem cell frequency, we quantified stem cells in BXD recombinant inbred mice (offspring of C57BL/6 and DBA/2). The resulting strain distribution pattern showed high concordance with a marker that mapped to chromosome 18 (19 cM). Linkage with this genomic interval was associated with a likelihood of odds score of 3.3, surpassing the level required for significance. Interestingly, this segment, containing the EGR-1 gene, shows synteny with human chromosome 5q, a region strongly associated with various hematological malignancies. Our findings indicate that a gene mapping to this region is mutated in either C57BL/6 or DBA/2 (and possibly AKR) mice. These studies in apparently healthy mice may facilitate the identification of a gene implicated in human 5q-syndromes.

A population of murine hematopoietic progenitors expresses an endogenous retroviral gp70 linked to the Rmcf gene and associated with resistance to erythroleukemia.

Multiple copies of retroviral sequences are stably integrated in the genomes of many higher organisms, and are thus transmitted vertically to offspring via the germline (1). Most of these heritable viral genes are not expressed, and expression, when observed, is commonly limited to envelope (env) genes as demonstrated by the presence of cell surface and serum envelope glycoprotein (gp70) in mice. Studies of the mouse have shown that certain tissues such as the reproductive tract and lymphoid organs are common sites for the expression of endogenous env genes, suggesting that the transcription of at least some endogenous sequences is tissue specific. The transcription of endogenous viral genes is regulated by both cis and trans mechanisms (2-5) and their expression can be temporally linked to differentiation and development (6-8). The consequences to the host of endogenous retroviral genes are varied. At one extreme, expression of endogenous virus can result in the development of leukemia and death. Another potentially detrimental effect is that of insertional mutagenesis, seen when the integration of retroviral sequences interrupts the functioning of a cellular gene (9, 10). However, it is now clear that expression of endogenous retroviral genes may also have a beneficial effect for the host: namely, mediating resistance to retroviral leukemias as has been demonstrated for the Fv-4 gene in mice (11) and some ea loci in chickens (12). This form of resistance is due to the blockage of cellular viral receptors by the expression of envelope glycoprotein on the cell surface. The Rmcf locus of the mouse is another resistance gene that may exert its effect by the expression of an endogenous env gene. A summary of our current state of knowledge concerning the Rmcf gene is shown in Table I. The Rmcf gene was originally described when it was observed that fibroblast cell cultures derived from certain strains of mice restricted the replication of recombinant mink cell focus-forming(MCF)1 viruses (13). As detailed in Table I, DBA/2 mice are the prototypic strain exhibiting the Rmcf resistance (Rmcf(r)) phenotype. Cell cultures from other strains, such as C57BL/6 and IRW, are permissive for MCF viral replication and are termed Rmcf sensitive (Rmcf(s)). Previously, we described two allelic forms of an endogenous env gene, whose expression is linked to the Rmcf gene (14). Cell cultures from Rmcf(r) mice express gp70 related to that of MCF viruses, whereas cultures derived from Rmcf(s) mice either express no gp70 (IRW) or express an endogenous xenotropic gp70 (C57BL/6). These two gp70 alleles are detectable by type-specific mAbs.

Genotype-restricted growth and aging patterns in hematopoietic stem cell populations of allophenic mice.

We have studied contributions to hematopoiesis of genetically distinct stem cell populations in allophenic mice. Chimeras were made by aggregating embryos of inbred strains known to differ with respect to stem cell population kinetics. One partner strain (DBA/2) has previously been shown to normally have a stem cell (CFU-S) population of which 24% are in S-phase of the cell cycle, whereas the homologous population of the other partner strain (C57BL/6) was characterized by having only 2.6% in cycle (7). Contributions of the chimeric stem cell population to mature blood cell pools were studied throughout the life of the mice and intrinsic differences in stem cell function and aging were reflected in dynamic patterns of blood cell composition. The DBA/2 stem cell population was eclipsed by stem cells of the C57BL/6 genotype and, after 1.5-3 yr, the hemato-lymphoid composition of 22 of 27 mice studied for this long had shifted by at least 25 percentage points toward the C57BL/6 genotype. 8 of the 27 had hematolymphoid populations solely of C57BL/6 origin. To test whether or not a population of stem cells with an inherently higher cycling rate (DBA/2) might have a competitive advantage during repopulation, we engrafted allophenic marrow into lethally irradiated (C57BL/6 x DBA/2)F1 recipients. DBA/2 hematopoiesis was predominant early, far outstripping its representation in the marrow graft. Perhaps as a consequence of inherently greater DBA/2 stem cell proliferation, the populations of developmentally more restricted precursor populations (CFU-E, BFU-E, CFU-GM, CFU-GEMM) showed an overwhelming DBA/2 bias in the first 2-3 mo after engraftment. However, as in the allophenic mice themselves during the aging process, the C57BL/6 genotypic representation was ascendant over the subsequent months. The shift toward C57BL/6 genotype was first documented in the marrow and spleen precursor cell populations and was subsequently reflected in the circulating, mature blood cells. Bone marrow-derived stromal cell cultures from engrafted mice were studied and genotypic analyses showed donor representation in stromal cell populations that reflected donor hematopoietic contributions in the same recipient. Results from these studies involving two in vivo settings (allophenic mice and engraftment by allophenic marrow) are consistent with the notion that a cell autonomous difference in stem cell proliferation confers on one population a competitive repopulating advantage, but at the expense of longevity.

Erythropoietin causes down regulation of colony-stimulating factor (CSF-1) receptors on peritoneal exudate macrophages of the mouse.

We have shown that erythropoietin (epo), the primary regulator of erythrocyte formation, diminished the binding to peritoneal exudate macrophages (PEM) of the principal macrophage growth regulator, colony-stimulating factor (CSF-1). The effect of epo on 125I-CSF-1 binding was dose-dependent; at a concentration of 1-2 U of epo/ml (10(-10) M), CSF-1 binding was almost completely suppressed. Erythropoietin did not compete with CSF-1 for occupancy of the latter's receptors. The effect of epo on CSF-1 binding occurred at 37 degrees C but not at 2 degrees C, and during the continuous exposure of PEM to epo at 37 degrees C we found that CSF-1 binding reached a nadir at 1 h and recovered to pre-exposure levels in 7 h. Our novel results are consistent with the notion that specific receptors for epo exist on the cell surface of PEM and that binding of epo sets in motion a series of cellular events resulting in the internalization of CSF-1 receptors. Thus epo causes down regulation of CSF-1 receptors on PEM. We have previously shown that epo causes suppression of CSF-induced granulocyte-macrophage colony formation by mouse bone marrow cells. The results we present here provide a possible mechanism for these results.

Simultaneous effects of erythropoietin and colony-stimulating factor on bone marrow cells.

Erythropoietin or colony-stimulating factor, or both, were added to rat or mouse marrow cell cultures, and the responses to each inducer were measured. Colony-stimulating factor caused the suppression of erythropoietin-stimulated hemoglobin synthesis, and erythropoietin caused the suppression of the granulocyte-macrophage colony formation that is dependent on colony-stimulating factor. The extent of suppression by each inducer was dose-dependent. Marrow cells from plethoric rats were more sensitive to suppression of erythropoietin action by colony-stimulating factor than were normal marrow cells. These findings suggest that either (i) the receptors for erythropoietin and for colony-stimulating factor have overlapping specificities and that the "wrong" inducer may bind without having an inductive effect, or (ii) the target cells for erythropoietin and colony-stimulating factor are very closely related or are the same.

Unraveling the complex regulation of stem cells: implications for aging and cancer.

Substantial progress in embryonic and adult stem cell research in the past several years has yielded a wealth of information regarding the mechanisms regulating self-renewal and differentiation, two processes often used to define stem cells. Recent evidence suggests that epigenetic as well as genetic processes maintain stem cells in a pluripotent state as well as dictate their transition to more restricted stages of development. In this review, we discuss two emerging themes in stem cell biology, epigenetic control of gene expression and post-transcriptional regulation via microRNAs. We summarize how these regulatory mechanisms facilitate various aspects of normal stem cell biology and extend the discussion to their involvement in aging and tumorigeneisis, two biological phenomena intimately tied to stem cells. We speculate that aberrant epigenetic events and altered miRNA expression profiles in aged stem cell populations play important roles in carcinogenesis.

The isoflavone genistein inhibits LPS-stimulated TNFalpha, but not IL-6 expression in monocytes from hemodialysis patients and healthy subjects.

BACKGROUND: Whole blood and peripheral blood mononuclear cells from hemodialysis (HD) patients show increased production and secretion of inflammatory cytokines. We determined the contribution of blood monocytes to the production of inflammatory cytokines in whole blood from HD patients. METHODS: Whole blood and isolated mononuclear cells from HD patients and healthy control subjects were preincubated with the isoflavone genistein and stimulated with LPS. TNFalpha, IL-6 and IL-10 formation in the whole blood was measured with ELISA and intracellular cytokine formation in CD 14-positive monocytes was determined by flow cytometry. RESULTS: Unstimulated blood levels of TNFalpha, IL-6 and IL-10 were significantly elevated in HD patients compared to controls, but intracellular monocyte content of these cytokines was identical between groups. LPS induced a robust TNFalpha response in both whole blood and monocytes, and TNFalpha formation was 2.3-fold higher in blood from HD patients compared to controls. A similar trend was observed in monocytes. Conversely, LPS stimulation increased IL-6 levels >1000-fold in whole blood, albeit without a noticeable difference between groups. Only minor increases in monocyte IL-6 content were observed. The isoflavone genistein did not inhibit IL-6 formation and did not alter basal TNFalpha levels, but genistein selectively blocked LPS-induced TNFalpha formation in whole blood and monocytes from both groups. CONCLUSION: Intracellular levels of TNFalpha, IL-6 and IL-10 in monocytes are indistinguishable between HD patients and healthy controls. However, monocytes from HD patients are selectively primed for enhanced TNFalpha secretion in response to LPS. The selective inhibition of monocyte TNFalpha production by genistein may explain the anti-inflammatory action of this phytochemical observed in vivo.

Resistance to erythroleukemia in immunodeficient xid- CBA/N mice with susceptibility after immune stimulation.

CBA/N (xid-) mice failed to develop erythroleukemia when inoculated with an NB-tropic, anemia-causing Friend virus stock (FVA), while Fv-2ss mouse strains succumbed rapidly to the characteristic Friend disease, even after a virus dose 30-fold lower than that given to CBA/N mice. Immunization with bacterial antigens or with spleen cell allografts prior to FVA inoculation rendered CBA/N mice highly susceptible to FVA. Transplantation studies confirmed that non-immunized CBA/N mice were able to both support erythroleukemic cells and permit erythroleukemic transformation, thus arguing against host defense mechanisms as a cause of resistance. On the basis of early erythroid progenitor cell sensitivity to hydroxyurea in vivo, the CBA/N strain appeared to have the FVA sensitive genotype (Fv-2ss). These results imply that CBA/N mice are not intrinsically resistant to FVA and that an as yet unknown type of immunological activity, evoked both by various immunizations and allogeneic transplantation, is required for Friend leukemogenesis in this immunodeficient inbred strain. These findings further suggest that the erythroid target cells transformed by Friend viruses are influenced by immunological activity.

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.

Amphotericin-B and monensin potentiation of murine erythropoiesis in vitro: a possible role for sodium ions.

To study the role of monovalent cation flux in erythropoiesis we cultured mouse bone marrow cells with amphotericin B (AmB), monensin, valinomycin, or Etruscomycin. At low doses the polyene antibiotic AmB has been shown to increase cell permeability to Na+ and K+ and we found that it potentiated erythropoietin (epo)-stimulated erythroid-colony (CFU-E) and burst (BFU-E) growth at concentrations ranging from 0.5-1.0 micrograms/ml. Monensin, a sodium-specific ionophore, potentiated epo-stimulated erythroid growth at concentrations of 1-30 nM. On the other hand, a potassium-specific ionophore, valinomycin, did not cause potentiation, but rather suppressed epo-dependent colony formation. Etruscomycin, another polyene, but one which in mammalian cells increases ion permeability only at toxic concentrations, was also suppressive. Potentiating concentrations of AmB and monensin increased the sensitivity of CFU-E and BFU-E to epo and at saturating epo levels increased the numbers of erythroid colonies and bursts by about 40%. Neither AmB nor monensin stimulated erythroid growth in the absence of epo. We found a 20-fold difference in the AmB concentrations comprising the maximally potentiating dose in C57BL/6 and AKR marrow cultures. This is consistent with observed differences between these two mouse strains with regard to other effects of AmB on them, including the immunoadjuvant properties of AmB. Our results showing potentiation due to sodium ion flux may be related to previous work showing potentiation of erythroid differentiation caused by calcium ion flux, since sodium ion movement may directly affect the intracellular calcium ion concentration.

Differentiation of chimeric bone marrow in vivo reveals genotype-restricted contributions to hematopoiesis.

Bone marrow transplantation is of increasing utility in cancer treatment and is an important component of gene therapy protocols. Understanding the functional identities of progenitor cells involved in repopulation is important for the optimal application of this procedure. We have simultaneously used two types of genetic markers to study engraftment of mice after irradiation. The first involves intrinsic genetic differences, including a cellular marker, between two mouse strains used to construct chimeric mice by aggregating embryos. To produce a second marking system, bone marrow from these allophenic mice was subsequently infected with retrovirus. Individual progenitor cells, including primitive lympho-hematopoietic stem cells, participating in repopulation were identified by virtue of their uniquely marked clonal progeny. In this way numbers and genotypic identities of clones contributing to repopulation were determined. Engraftment could be divided into two distinguishable temporal phases. The first comprised roughly the first 3-4 months following transplant and was characterized by numerous clones, many of which apparently had limited lineage potencies. The subsequent phase was characterized by few, often a single, clones represented in all lympho-hematopoietic tissues. These findings are consistent with the notion that different classes of progenitor cells are differentially responsible for temporal progression. More differentiated, perhaps lineage restricted, progenitors transiently dominate the first few months before the emergence of pluripotent stem cell clones. Senescence of progenitors of the first phase may reflect their limited lifespans. A clear genotypic difference was obvious in engraftment. Cells of one strain, DBA/2, completely dominated the first temporal phase, whereas the C57BL/6 partner strain dominated the second phase. The genotype-restricted dominance of different stages of repopulation suggests important differences in the organization and regulation of stem and progenitor cell populations. Inherent differences in seeding, proliferation, and differentiation of progenitors of the two inbred strains may account for the differences. This in vivo model of competitive repopulation provides the opportunity to explore potentially important loci in the process of engraftment. We propose that DBA/2 progenitor cells, due to a proliferative and/or numerical advantage, account for their superiority immediately after engraftment. C57BL/6 stem cells, with long-term repopulating potential, predominate later, perhaps because of subtle numerical or proliferative advantages.

Quantitation of erythroid differentiation in vitro using a sensitive colorimetric assay for hemoglobin.

We have developed a new colorimetric method to detect early erythroid clonogenic progenitor cells by virtue of their hemoglobin-containing progeny in vitro. The method relies upon the specific oxidation of 2,7-diaminofluorene (DAF) by the pseudoperoxidase activity of hemoglobin (Hb). Generation of fluorene blue (FB), the chromophore generated upon oxidation of DAF, was a linear function of Hb and erythrocyte concentration. Measurement of FB absorbance at 610 nm was 80 times more sensitive than measurement of Hb absorbance at 530 nm. The limit of resolution was approximately 8 pmol of Hb corresponding to the Hb content of approximately 5000 murine peripheral blood erythrocytes. FB concentration closely paralleled eight-day BFU-E colony formation as a function of erythropoietin dose. However, the method was unable to detect a response for CFU-E to erythropoietin because of persistently high background levels of Hb in two-day cultures. Generation of FB by cells likely to contain myeloperoxidase was negligible, and FB production was not correlated with the number of CFU-GM colonies. DAF oxidation by hemoglobin showed a higher hydrogen peroxide optimum than oxidation by microperoxidase, so that, under the conditions used, FB generation by peroxidase was not favored. As a further confirmation of specificity, peritoneal exudate cells failed to show significant generation of FB. The DAF reagent was also used to stain both CFU-E and BFU-E colonies in situ, and was more sensitive than benzidine dihydrochloride in the histochemical detection of Hb. DAF can replace hazardous benzidine-related compounds for the staining erythroid colonies in culture dishes, and the method offers a new quantitative approach to the study of erythropoietin responsiveness in vitro.

Stem cell quiescence/activation is reversible by serial transplantation and is independent of stromal cell genotype in mouse aggregation chimeras.

We made use of a previously described in vivo model of chimeric mice created by embryo aggregation that allows the study of contributions to both lymphohematopoiesis and marrow stroma by two genotypically distinct cell populations. Day-2 embryos from C57BL/6 and DBA/2 strains were fused to produce allophenic chimeric mice that proved to have contributions from each strain in all the tissues of the body and that permitted study of competitive contributions to blood formation. Although the contribution of DBA/2 stem cells to hematopoiesis gradually ceased in an age-related manner so that all blood cells in aged chimeras were C57BL/6 in origin, here we show that, in contrast, the extent of stromal chimerism, determined by the fibroblast colony-forming unit (CFU-F) assay, was maintained and was remarkably uniform from one marrow site to another. This result is consistent with a polyclonal nonhematopoietic origin of the CFU-F and suggests that the decline in DBA/2 blood cells was not dependent on similar changes in genotype-matched stroma, but was instead an intrinsic property of this stem cell population. These intrinsic stem cell properties were further examined by serial bone marrow transplantation. When marrow from a chimera with no detectable DBA/2 blood cells was transplanted into irradiated recipients, cells of DBA/2 genotype significantly contributed to early hematopoietic engraftment, demonstrating that the DBA/2 stem cell population was not extinguished in the chimeric donor, but rather had entered a reversible state of quiescence. Reactivation of the DBA/2 stem cell population, however, was short-lived, and long-term engraftment of recipients was accomplished by donor cells of the partner strain (C57BL/6). However, transient reactivation of the quiescent (DBA/2) stem cell pool again occurred with a second round of transplantation. These surprising results demonstrate, for the first time, selective and reiterated inactivation and reactivation of a stem cell population depending on hematopoietic needs. Moreover, the results suggest that genetic differences in the stem cell populations of coexistent strains account for the selective responses described.

Autonomous behavior of hematopoietic stem cells.

Mechanisms that affect the function of primitive hematopoietic stem cells with long-term proliferative potential remain largely unknown. Here we assessed whether properties of stem cells are cell-extrinsically or cell-autonomously regulated. We developed a model in which two genetically and phenotypically distinct stem cell populations coexist in a single animal. Chimeric mice were produced by transplanting irradiated B6D2F1 (BDF1) recipients with mixtures of DBA/2 (D2) and C57BL/6 (B6) day-14 fetal liver cells. We determined the mobilization potential, proliferation, and frequency of D2 and B6 stem and progenitor cells in animals with chimeric hematopoiesis. After granulocyte colony-stimulating factor (G-CSF) administration, peripheral blood D2 colony-forming units granulocyte-macrophage were fourfold to eightfold more numerous than B6 progenitors. We determined that D2 and B6 progenitors maintained their genotype-specific cycling activity in BDF1 recipients. Chimeric marrow was harvested and D2 and B6 cell populations were separated by flow cytometry. Cobblestone area-forming cell (CAFC) analysis of sorted marrow showed that the number of late appearing CAFC subsets within the D2 cell population was approximately threefold higher than within the B6 fraction. We performed secondary transplantation using unfractionated chimeric marrow, which was given in limiting doses to lethally irradiated BDF1 recipients. Comparison of the proportion of animals possessing D2 and/or B6 leukocytes 5 months after transplant revealed that the frequency of D2 LTRA was approximately 10-fold higher than B6 LTRA numbers. Our data demonstrate that genetically distinct stem cell populations, coexisting in individual animals, independently maintain their parental phenotypes, indicating that stem cell properties are predominantly regulated cell-autonomously.

Selective elimination (purging) of contaminating malignant cells from hematopoietic stem cell autografts using recombinant adenovirus.

In this work we have explored the use of adenoviral vectors for the purging of cancer cells from hematopoietic stem cell (HSC) autografts. We showed that a recombinant adenovirus expressing the herpes simplex-1 thymidine kinase gene (AD-tk) plus ganciclovir (GCV) killed HELA cells more effectively than did GCV alone. HELA cells were then mixed with human HSCs and exposed to AD-tk/GCV. AD-tk/GCV reduced the number of HELA colonies to 4% of control values, with no detectable reduction in the hematopoietic progenitor, colony forming unit-granulocyte/monocyte (CFU-GM). Similar studies of the JB6 non-Hodgkins lymphoma cell line showed a reduction to 5% of controls; studies of MCF-7, a breast carcinoma cell line, showed a reduction to 30% of controls, with no CFU-GM toxicity. Thus, AD-tk mediated selective killing of contaminating tumor cells. We also evaluated a recombinant adenovirus encoding the tumor suppressor gene p53 (AD-p53). AD-p53 was able to selectively kill all three cell lines (reducing tumor colonies approximately 100-fold) without any toxicity to CFU-GM. Although both AD-tk/GCV and AD-p53 were effective in these experiments, AD-p53 seemed to be more potent. Adenoviral vectors show promise for selectively targeting cancer cells that contaminate HSC autografts.

Does functional depletion of stem cells drive aging?

The regenerative power of stem cells has raised issues about their relation to aging. We focus on the question of whether a decline in the function of stem cells may itself be a significant feature of aging. The question is implicitly two-fold: does functional depletion of stem cells affect the accumulation of aging-related deficits, and--whether or not depletion is significant--can activation of stem cells alleviate deficits? Two types of system are considered: 1) the exhaustible pool of ovarian follicles. The depletion of follicles leads to the aging-related phenomenon of menopause; and 2) the reserve of hematopoietic stem cells. Substantial numbers are sustained throughout life, but in mouse models, endogenous replicative activity has been shown to decline sharply with age. We discuss the possible implications of these observations for the rate of aging and the prospects for intervention.

Genetic control of lifespan: studies from animal models.

The premise that there are genes that wield a strong influence on longevity has, until recently, not been a popular one and there has been no concerted effort to find such genes. However, the finding that single genes can have large effects on the lifespans of yeast, worms and flies raises the possibility that individual genes in mammals may similarly have relatively large effects on longevity. Recent advances in mammalian genetics, many associated with the large-scale efforts to sequence the human and mouse genomes, have accelerated the search for longevity genes, principally in mice. Here, we review results using animal models that have recently shed light on genes regulating longevity and ageing patterns. A large number of genetically defined strains of mice are available and this, together with their established history of use in genetic research and their relatively short lifespans, has made murine models particularly useful. We also review our own work in which genes regulating mouse lifespan and those regulating cell cycling of haematopoietic progenitor cells have been mapped to the same locations in the genome. These results suggest that some of the same genes affect both traits, and further suggest a cause-and-effect relationship between cumulative cell-cycle activity and longevity of an organism.