Lifelong hematopoiesis in both reconstituted and sublethally irradiated mice is provided by multiple sequentially recruited stem cells.

OBJECTIVE: To evaluate the dynamics of stem cell production to hematopoiesis, the number of active stem cell clones and the lifespan of individual clones were studied. MATERIALS AND METHODS: The clonal contribution of primitive hematopoietic stem cells (HSC) responsible for long-term hematopoiesis was determined using two approaches. In one model, irradiated female mice were reconstituted with retrovirally marked male hematopoietic cells. In the second model, mice were irradiated sublethally without hematopoietic cell transplantation. In both models, bone marrow cells were serially sampled from the same mouse throughout a 12- to 20-month period and injected into irradiated recipients for analysis of day 10 colony-forming unit-spleen (CFU-S). The donor origin of CFU-S was determined by the presence of retrovirally marked cells or cells with chromosomal aberrations. RESULTS: The results of the two essentially different models show that 1) hematopoiesis is mainly the product of small clones of hematopoietic cells; 2) the lifespan of the majority of clones is only 1 to 2 months; 3) the clones usually function locally; and 4) the vast majority of the clones replace one another sequentially. Primitive HSCs capable of producing long-lived clones (about 10% among all clones), which exist during the entire life of a mouse, were detected by the radiation-marker technique only. CONCLUSION: Multiple short-living clones (at least on the level of CFU-S production) comprise the vast majority of the active stem cells in transplanted recipients or after endogenous recovery from sublethal irradiation.

Local clonal analysis of the hematopoietic system shows that multiple small short-living clones maintain life-long hematopoiesis in reconstituted mice.

We describe here a technique to study the clonal contribution of primitive stem cells that account for long-term hematopoiesis in the same mouse over a 14-month period. Specifically, irradiated recipient female mice were transplanted with retrovirally marked male hematopoietic progenitors. Bone marrow was then collected repeatedly from local sites from the same mice throughout a 14-month period and injected into secondary irradiated recipients for analysis of donor retrovirally marked day-11 colony-forming unit-spleen (CFU-S-11). We have tracked the temporal in vivo fate of 194 individual CFU-S-derived cell clones in 38 mice reconstituted with such retrovirally marked bone marrow cells. Our data show that long-term hematopoiesis is maintained by a large number of simultaneously functioning small, shortlived (1 to 3 months) clones that usually grow locally with little or no dispersion between different regions of the hematopoietic system. Furthermore, the clones that disappeared were never detected again. The data suggest that normal hematopoiesis is supported by the sequential recruitment of marrow repopulating cells into a differentiation mode.

Transfer of human adenine deaminase gene into murine hematopoietic stem cells: sequential study of spleen colony-forming units from bone marrow of living mice and the requirement of the microenvironment.

Irradiated female mice were reconstituted with male hematopoietic stem cells (HSCs) retrovirally marked with human adenine deaminase (hADA) complimentary DNA. HSCs were incubated with interleukin-6 and stem cell factor before coculture with GP+E86-producing cells. Bone marrow HSCs were infused intravenously to irradiated mice and spleen colony-forming units (CFU-S) were evaluated for hADA marked clones by Southern blot analysis. 45 of 54 CFU-S were marked by the hADA gene sequence with multiple copies integrated per genome. Oligoclonal hematopoiesis evolved over time with 1-2 clones demonstrated 5-11 months after reconstitution. Comparable results were obtained with embryonic fetal liver HSCs. Incubation of bone marrow HSCs with adherent stromal cells rather than growth factors produced less efficient gene transfer, and polyclonal hematopoiesis was not observed. Donor origin was established by the Y chromosome probe. These results support the clonal succession model of hematopoiesis.

Additive effect of erythropoietin and heme on murine hematopoietic recovery after azidothymidine treatment.

The ability of combination treatment with erythropoietin (Epo) and heme to rescue hematopoietic activity in mice from the suppressive effect of azidothymidine (AZT) was determined. Exposure of mice to AZT for 5 weeks produced marked anemia, thrombocytopenia, neutropenia, and weight loss, whereas mice that received Epo and heme for 3 subsequent weeks showed significant alleviation of AZT cytotoxicity. Treatment with Epo (10 U for 5 times/week) stimulated hematopoietic recovery in the AZT-treated animals and reduced the severe anemia and thrombocytopenia by 3 weeks. Administration of a lower Epo dose (1 U Epo) resulted in only a modest retardation of AZT-induced anemia, although, when combined with heme, there was a great improvement in recovery of erythropoiesis. The combination of heme with Epo (10 U) produced the optimum response, resulting in almost normal recovery of bone marrow cellularity as well as recovery of burst-forming units-erythroid (BFU-E) and splenic hematopoietic progenitor content (colony-forming unit-spleen [CFU-S]) by the end of 3 weeks of post-AZT treatment. Treatment with heme alone markedly enhanced the recovery of BFU-E and CFU-S, as well as body weight post-AZT; however, this recovery was not to the extent seen in combination with Epo (10 U). Long-term bone marrow cultures (LTBMCs) established from mice exposed to AZT for 8 weeks showed a marked reduction in cellularity and this was completely alleviated when mice received heme and Epo (10 U) for 3 weeks after 5 weeks of AZT administration. The additive effect of heme and Epo was seen in BFU-E production, as well as in CFU-S production, in LTBMCs. Thus, heme exerts a significant protective effect on hematopoietic progenitors in vivo and may be of potential clinical use in combination with Epo to promote effective erythropoiesis in the setting of AZT therapy.

Synergistic effect of heme and IL-1 on hematopoietic stromal regeneration after radiation.

Results from this study show that a combination of heme and interleukin-1 (IL-1) treatment resulted in the most improved recovery of hematopoietic-stromal regeneration after sublethal irradiation. Less pronounced effects were obtained when heme or IL-1 were given singly. Sublethal irradiation of mice produced an initial (as early as day 1) intense depression of the hematopoietic system as evidenced by leukopenia. In vivo treatment of animals with heme in combination with IL-1, accelerated hematopoietic and stromal regeneration as determined by hematopoietic spleen colony forming unit assay (CFU-S), erythroid (BFU-E), myeloid (CFU-GM) clonal cultures, long-term bone marrow cultures (LTBMC), and the ability to regenerate hematopoiesis by ectopic (renal) stromal hemopoietic transplantation. Sixteen days after irradiation, leukocyte levels in heme and IL-1 treatment groups were higher than non-treated animals and were near normal values by 27 days. One day after irradiation, the capacity of stromal progenitors to form new bone and hematopoietic cells (ectopic foci) was severely impaired, but recovered after 2-4 weeks. This recovery process was accelerated in heme and IL-1-treated animals. BFU-E, CFU-GM, and CFU-S capacity was also severely impaired in all animals 1-27 days after irradiation. CFU-S was only 0.15% of control by day 1 and 5% of control by day 16. Treatment with heme or IL-1 improved recovery by as much as 70% after 27 days of irradiation. A similar but enhanced recovery was seen for BFU-E and CFU-GM, with erythroid recovery the best. Total cellularity, adherent cell layer (ACL) formation, and clonogenic capacity by LTBMCs (10 weeks) derived from irradiated animals was severely reduced, whereas the hematopoietic capacity by LTBMCs derived from heme- and IL-1-treated animals had recovery values similar to non-irradiated controls. These results suggest therapeutic use of heme and IL-1 after chemotherapy or bone marrow depression may be beneficial.

The hematopoietic stromal microenvironment promotes retrovirus-mediated gene transfer into hematopoietic stem cells.

In this study we report on the establishment of novel conditions which permit efficient retrovirus-mediated gene transfer of human adenosine deaminase (ADA) into murine hematopoietic progenitors. Using Southern blot analysis and an ADA probe, we demonstrated that prestimulation of bone marrow cells over an in vitro culture of adherent stromal cell layers (ACLs) for two days provides favorable conditions for gene transfer in the absence of exogenous growth factors. In bone marrow transplant recipients reconstituted with retrovirally-marked cells, ADA was detected in spleen, thymus and bone marrow cells of the recipients eight months after transplantation. These observations were also seen in transplants of embryonal hematopoietic stem cells. By using different incubation protocols, it was found that the developmental fate of hematopoietic stem cells varied with the presence of exogenous growth factors or an ACL in the prestimulation phase. Polyclonal hematopoiesis with multiple clones appearing simultaneously was revealed in mice reconstituted with growth factor-stimulated cells four months after transplantation. This was detected by multiple integration patterns of ADA integration into the genomes of individual colony forming units-spleen (CFU-S) in transplantation recipient mice. In contrast, two to five months after transplantation, polyclonal hematopoiesis was not observed in mice reconstituted with cells infected in the absence of growth factors. It appears that utilization of the bone marrow microenvironment through the use of an ACL results in a narrower spectrum of integration patterns, suggesting that a type of oligoclonal or monoclonal hematopoiesis is occurring. These studies demonstrate that an ACL provides novel conditions for successful gene transfer and stable integration of the vector into the genome. Use of an ACL may be advantageous for successful hematopoietic stem cell gene therapy.

Long-term bone marrow stromal and hemopoietic toxicity to AZT: protective role of heme and IL-1.

We studied the immediate and long-term effects of azidothymidine (AZT) and heme on murine hemopoietic and stromal progenitor cells in vivo and in vitro. Treatment of mice for 37 days with AZT produced anemia and leukopenia, whereas combined treatment with heme abrogated some of the toxic effects which were apparent even 2 weeks after cessation of treatment. Quantitation of spleen (CFU-S), erythroid (BFU-E) and myeloid (CFU-GM) colony formation from AZT-exposed animals revealed reductions in these progenitors, and this was partially reversed after heme treatment, especially when mice were allowed a 2-week recovery period. Long-term bone marrow cultures (LTBMC) of cells from treated groups revealed difficulty in establishing an adherent cell layer (ACL) by the first week in culture. Total cellularity, CFU-S, BFU-E and CFU-GM clonogenic potential of cultures remained depressed throughout 10 weeks of culture, whereas heme treatment overcame these depressions when AZT-exposed mice were allowed to recover for 14 days prior to culture of their cells in LTBMC. Interleukin-1 (IL-1) treatment to the same recovery group of AZT-exposed mice also resulted in an improvement of CFU-GM growth in LTBMC that was not seen in the nonrecovered group. Transplantation of cells from treated mice under the renal capsule of recipient mice revealed that AZT depressed the regeneration of osteogenic and hemopoietic cell growth within ectopic foci. These effects were reversed with heme treatment in vivo. In other experiments, heme was found to inhibit human immunodeficiency virus (HIV-1) reverse transcriptase and to potentiate the activity of AZT triphosphate against HIV-1 reverse transcriptase. In summary, these results demonstrate that AZT inhibits the growth and development of a variety of hemopoietic, stromal and adherent cells in vivo and in vitro. Treatment of animals with heme produced recovery to near normal levels and suggests possible therapeutic potential.

Hematopoietic effects of benzene inhalation assessed by murine long-term bone marrow culture.

The strong and long-lasting hematotoxic effect after benzene exposure in vivo (300 ppm, 6 hours/day, 5 days/week for 2 weeks) was assessed in mice with bone marrow cells grown in long-term bone marrow culture (LTBMC). Bone marrow cultures initiated 1 day after the last benzene exposure did not produce adequate numbers of hematopoietic cells over 3 weeks and, in most cases, no erythroid or myeloid clonogenic cells could be recovered. The adherent cell layer of these cultures had a lower capacity for supporting in vitro hematopoiesis after the second seeding with normal bone marrow cells compared with control cultures. Two weeks after the last benzene exposure, body weight, hematocrit, bone marrow cellularity, and committed hematopoietic progenitor content (BFU-E and CFU-GM) were regenerated to normal or subnormal values, whereas hematopoiesis in LTBMC was very poor. Over 8 weeks, little or no significant committed progenitor production was observed. Treatment of mice exposed to benzene with hemin (three doses of 3 micrograms/gm body wt, iv, over 2-week for a total dose 9 micrograms/gm) partially overcame the toxic effect of benzene on the hematopoietic system as measured by the LTBMC method. Cultures from mice treated with hemin had modest recovery of BFU-E and CFU-GM clonogenic potential after 5 to 6 weeks in LTBMC. In contrast, little or no recovery was obtained for the adherent cell layer clonogenic capacity, even after hemin treatment. These results clearly indicate a strong, long-lasting toxic effect on the bone marrow stroma and a limited recovery of hematopoietic potential by clonogenic cells of the nonadherent population after in vivo hemin treatment.

Gene therapy model for stromal precursor cells of hematopoietic microenvironment.

Marker bacterial Neor gene was transduced by retroviral gene transfer into stromal precursor cells making up the hematopoietic microenvironment in murine long-term bone marrow cultures (LTBMC). Cultures were infected six times during the first 3 weeks of cultivation. At 4 weeks, the adherent cell layers (ACLs) were implanted under the renal capsule of syngeneic unirradiated and irradiated mice. Cells from newly formed ectopic foci were explanted into secondary LTBMC. ACLs containing the marker gene were detected by polymerase chain reaction. About 74% of stromal cells in ACLs contained Neor gene. The possibility of stable gene transduction into stromal precursor cells competent to transfer the hematopoietic microenvironment was established.

Hemin stimulation of hemopoiesis in murine long-term bone marrow culture.

The effect of various concentrations of exogenous hemin on cellularity and hemopoietic clonal potential of cells maintained in murine long-term marrow cultures (LTBMC) was studied. Hemin, at concentrations of 1 and 10 microM, was added weekly to LTBMC and was found to produce a significant increase in cellularity for up to 8 weeks in culture. Lower concentrations of hemin (0.1 microM) were more effective for sustained cellularity in older cultures (10-12 weeks). Prior exposure of the adherent cell layer to high concentrations of hemin (10 microM) was found to have a beneficial effect on the support of newly seeded cultures; however, the effect of lower hemin concentrations (0.1-1 microM) on stromal cell layer formation was not significant. Supplementation of hemin for the first week in culture increased cumulative cell production as well as the number of granulocyte-macrophage colony-forming units (CFU-GM), and longevity of hemopoiesis in LTBMC was significantly increased with 0.1 microM hemin. In contrast with data obtained in short-term cultures, hemin in this system primarily affected the myeloid line of differentiation, whereas there was a less noticeable effect on the early erythroid progenitors (erythroid burst-forming units, BFU-E). Hemin, at 0.1 microM, increased spleen colony-forming units (CFU-S) to numbers several-fold higher than those of the control. Results suggest that hemin may produce mobilization of hemopoietic cells and committed precursors from adherent cells into suspension. Further, supplementation with hemin in LTBMC significantly increased the myeloid progenitor compartment and longevity of culture without altering the erythroid compartment.

Induction of hematopoietic microenvironment by the extracellular matrix from long-term bone marrow cultures.

Extracellular matrix (ECM) plays an important role in the regulation of hematopoiesis. The ECM obtained from murine long-term bone marrow cultures (LTBMCs) induces hematopoietic foci formation within 3 months after implantation under the murine renal capsule. The foci consist of approximately 3 x 10(6) hematopoietic cells and function for at least 11 months. The induced stroma contains transplantable precursors capable of transferring a hematopoietic microenvironment to secondary recipients, and is insensitive to the stroma-stimulating factor produced in recipient mice after irradiation. The ECM induces hematopoietic foci formation in chimeras irradiated by a dose which is lethal for most of the stromal precursors. These facts point to the differences observed between bone marrow stromal precursors and mesenchymal cells induced under the renal capsule. The foci contain bone, but its appearance is limited to early stages of foci growth, and depends on the dose of implanted ECM. Bone is not formed when the xenogeneic ECM from nonhematopoietic tissue is used as an inducer. In this case, the foci develop slowly and are observed only to the tenth month after implantation. The data obtained demonstrate a novel function of the ECM in the induction of a hematopoietic microenvironment.

Comparison of hemin enhancement of burst-forming units-erythroid clonal efficiency by progenitor cells from normal and HIV-infected patients.

The ability of peripheral-blood hematopoietic progenitor cells from AIDS patients and normal controls to respond to erythropoietin (Epo) was assessed for burst-forming units-erythroid (BFU-E). BFU-E colony formation from AIDS patients' peripheral blood responded to a wide range of Epo concentrations (0.5-4 U) in a similar manner as erythroid progenitors obtained from normal peripheral blood. The optimum dose response of BFU-E to Epo was 2 U which resulted in generation of 71 +/- 4 BFU-E in AIDS patients (n = 10), as compared to 77 +/- 5 BFU-E in normal donors (n = 3). The optimum concentration range of hemin enhancement of erythroid progenitor BFU-E was 10-50 microM. In all instances, Epo was essential for BFU-E growth. Inclusion of hemin at a concentration of 10 microM in AIDS patients' peripheral-blood erythroid progenitor cells resulted in enhancement of BFU-E by 136-215%. Similarly, inclusion of hemin (10-100 microM) in normal bone marrow erythroid progenitor cell cultures resulted in enhancement of BFU-E. Inclusion of an equivalent amount of iron or tin protoporphyrin to progenitors cells from AIDS patients' peripheral blood had no effect on the number of colonies observed. On the other hand, inclusion of another heme analogue, zinc protoporphyrin, in AIDS or normal cultures resulted in a 50% suppression of BFU-E colony formation. These results demonstrate that peripheral-blood mononuclear cells from AIDS patients retain the capacity to generate erythroid precursors such as BFU-E in the presence of Epo, and that hemin has a specific enhancement effect on growth of BFU-E colony formation obtained from peripheral blood or bone marrow cells.

Comparative effect of heme analogues on hematopoiesis in lymphoproliferative disorders.

Anemia is a common characteristic of lymphoproliferative disorders (LPD) and the impairment of blood formation in these disorders is not fully understood. Heme synthesis and the heme degradative enzyme heme oxygenase are critical to hematopoietic differentiation and disturbances may contribute to anemic states. Tin protoporphyrin (SnPP) is a potent inhibitor of heme oxygenase, and has proven to be a useful clinical agent. Bone marrow cells from seven patients with LPD were studied for their in vitro hemopoietic response to growth factors and SnPP. Heme oxygenase mRNA levels were determined by Northern blot analysis of bone marrow samples. Quantitation of hematopoiesis in cultures with erythropoietin or GM-CSF revealed adequate CFU-E, BFU-E and CFU-GM growth by LPD bone marrow. Inclusion of 10 µM SnPP in cultures was found to significantly enhance CFU-E/BFU-E growth by LPD marrows, whereas Zinc protoporphyrin had a marked inhibitory effect. Little or no effect by SnPP was seen on CFU-GM. In contrast, normal bone marrow cultures failed to show an enhanced response to 10 µM SnPP. Analysis of heme oxygenase mRNA levels revealed that LPD marrows had elevated expression of heme oxygenase mRNA as contrasted with normals. Furthermore, measurements revealed that heme oxygenase activity was markedly suppressed by SnPP in the LPD bone marrow cultures. Results lend further support to the importance of heme oxygenase in the differentiation process. Although LPD bone marrow cells may respond to erythropoietin in vitro, in stressed conditions where heme oxygenase is elevated, suppression of heme oxygenase may potentiate the erythropoietic response in this disease.

Individual clones of hemopoietic cells in murine long-term bone marrow culture.

Forty-seven individual hemopoietic cell clones bearing unique radiation markers were studied in long-term bone marrow cultures. Throughout cultivation clones appeared at different times, from 1 to 12 weeks after explantation, survived during 1-10 more weeks, and were characterized by marked variability in size. Usually, the number of metaphases peculiar to an individual clone rapidly increased, achieved maximum, and then underwent a decline. Cells of reliably disappearing clones were never seen again. The experimental results provide further evidence for the model of hemopoiesis by clonal succession.

Cells responsible for restoration of haemopoiesis in long-term murine bone marrow culture.

Long-term haemopoiesis in bone marrow culture is sustained by the progeny of haemopoietic progenitor cells (HPC), which differ from CFUs by very low sensitivity to repeated hydroxyurea (HU) injections. The transit time of a haemopoietic clone from HPC to cells proliferating in culture is 6-7 weeks. The results suggest that the stem cell continuum is an expansion type compartment, members of which gradually lose their proliferative potential during differentiation.

Origin of hemopoietic stromal progenitor cells in chimeras.

Intravenously injected bone marrow cells do not participate in the regeneration of hemopoietic stromal progenitors in irradiated mice, nor in the curetted parts of the recipient's marrow. The hemopoietic stromal progenitors in allogeneic chimeras are of recipient origin. The adherent cell layer (ACL) of long-term cultures of allogeneic chimera bone marrow contains only recipient hemopoietic stromal progenitors. However, in ectopic hemopoietic foci produced by marrow implantation under the renal capsule and repopulated by the recipient hemopoietic cells after irradiation and reconstitution by syngeneic hemopoietic cells, the stromal progenitors were of implant donor origin, as were stromal progenitors of the ACL in long-term cultures of hemopoietic cells from ectopic foci. Our results confirm that the stromal and hemopoietic progenitors differ in origin and that hemopoietic stromal progenitors are not transplantable by the intravenous route in mice.

Self-renewal capacity and clonal succession of haemopoietic stem cells in long-term bone marrow culture.

The CFU-s proliferative potential varied greatly during long-term cultivation. Most of the CFU-s in the cultures were represented by cells with low renewal capacity. Pre-CFU-s cells capable of producing multipotential colonies in methylcellulose, which contained CFU-s with a high proliferative potential, were identified in the culture. In cultivation of a mixture of cells of different karyotype their ratio changed rapidly from week to week. The findings were consistent with the hypothesis that haemopoietic stem cells are maintained in the culture by the products of a small number of clones which arise and decline in succession, and that pre-CFU-s, but not the CFU-s themselves, are clonogenic progenitors.