Long-term persistence of a nonintegrated lentiviral vector in mouse hematopoietic stem cells.

Objective: Lentiviral transduction is an established method for efficiently modifying the gene expression program of primary cells, but the ability of the introduced construct to persist as an episome has not been well studied.

Material And Methods: Here we investigated this issue in lethally irradiated female mice injected with 300 or 3000 doubly sorted male lin(neg), Sca-1(high), c-kit(high), Thy-1.1(low) mouse bone marrow cells that had been exposed in vitro to self-inactivating lentivirus vector encoding a green fluorescence protein (GFP) cDNA.

Developmental fate of hematopoietic stem cells: the study of individual hematopoietic clones at the level of antigen-responsive B lymphocytes.

We have shown previously that hematopoiesis in mice reconstituted with retrovirally marked hematopoietic stem cells (HSCs) is provided by multiple, mainly short-lived clones, as measured by retroviral insertion site analysis of individual spleen colony-forming unit (CFU-S)-derived colonies. However, the CFU-S is the relatively early progenitor and the contribution of each CFU-S in the steady-state hematopoiesis is uncertain. Here, we have studied the fate of individual mature B cells, as well as CFU-S, representing the progeny of retrovirally transduced marrow-repopulating cells (MRC).

Neoplastic transformation is not the cause of extremely long (more than 100 weeks) hematopoiesis maintenance of long-term bone marrow culture from TNF-deficient mice.

Total cell production and longevity of hematopoiesis in long-term bone marrow culture of tumor necrosis factor (TNF)-deficient mice (LTBM-TNFko) are increased. The rate of apoptosis is decreased during the first 40 weeks in culture, then the level of apoptosis reaches levels of wild-type cultures. Extended lifespan of primary cultures usually is the consequence of the neoplastic transformation.

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.

Long-term maintenance of hematopoiesis in irradiated mice by retrovirally transduced peripheral blood stem cells.

Mobilized peripheral blood stem cells (PBSC) are used as a source of hematopoietic stem cells for transplantation and gene therapy. It is still unclear, however, whether the PBSC are fully equivalent to normal bone marrow hematopoietic stem cells and whether they are able to provide long-term function of transgene in reconstituted mice. In the present study, mobilized PBSC from male mice were transduced with human adenosine desaminase gene (hADA) and were used for reconstitution of lethally irradiated female mice.

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).

Effect of cytokine treatment (granulocyte colony-stimulating factor and stem cell factor) on hematopoiesis and the circulating pool of hematopoietic stem cells in mice.

The mobilization of hematopoietic stem cells (HSCs) into the peripheral blood of mice was induced by recombinant human granulocyte colony-stimulating factor (rhG-CSF) (250 microgram/kg/d) alone or combined with recombinant rat stem cell factor (rrSCF) (34 microgram/kg/d), injected subcutaneously (s.c.) once a day for 10 and 17 days.

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.

Hematopoietic progenitor cell mobilization into the peripheral blood of mice using a combination of recombinant rat stem cell factor (rrSCF) and recombinant human granulocyte colony-stimulating factor (rhG-CSF).

A significant increase in the hematopoietic stem cell (HSC) concentration has been observed in the peripheral blood and spleen of mice treated with rhG-CSF alone or with a combination of rhG-CSF plus rrSCF. The longer the duration of cytokine treatment, the higher the stimulatory effect on stem cell mobilization. In addition, cytokine administration led to a reduced stem cell concentration in the bone marrow.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.