Hemonectin mediates adhesion of engrafted murine progenitors to a clonal bone marrow stromal cell line from Sl/Sld mice

Mutant Sl/Sld mice exhibit decreased marrow hematopoiesis. The defect is known to reside in the marrow microenvironment of these animals, which is reproduced in vitro by primary marrow explants as well as by cloned marrow stromal cell lines. Bone marrow progenitor cells are incapable of adhering to primary Sl/Sld stromal cells or cloned stromal cell lines derived from them to form cobblestone-islands and proliferate. The role of hemonectin, a marrow-specific adhesion protein in the defective hematopoiesis of the Sl/Sld mice, was studied. Indirect immunoperoxidase staining of marrow in situ from Sl/Sld mice showed little specific staining while specific staining was seen in a pericellular distribution in marrow from +/+ mice. Hemonectin expression in several cloned stromal cell lines from Sl/Sld mice was compared by immunoblotting with that in cloned stromal cell lines from normal +/+ littermates. Cell line Sld3, which has the least hematopoiesis supportive capacity in vitro, showed no detectable hemonectin by immunoblotting, while Sld1 and Sld2 showed detectable but greatly reduced amounts compared with normal +/+ 2.4, GBI/6, and D2XRII. Confluent cultures incubated with purified hemonectin and engrafted with enriched progenitors showed a significant increase in the cumulative number of cobbleston-islands and day 14 spleen colony-forming units (CFU-s) forming progenitors (39.15 +/- 3.6/dish; 16.3 +/- 3.1/dish, respectively), compared with untreated Sld3 cultures (cobblestone-islands 8.1 +/- 3.6/dish; CFU-s forming progenitors 8.8 +/- 0.05/dish). Hemonectin-mediated progenitor cell binding to the Sld3 stromal cells was specifically inhibited by antihemonectin but not by preimmune serum. These data support the role of hemonectin in early progenitor-stromal cell interactions.     

Stromal cells (CFU-F) in normal and genetically anemic mouse strains

Mice of the Sl/Sld genotype have an approximately 3-fold higher number of fibroblastoid progenitors (CFU-F) in their spleens than their normal +/+ littermates. Experiments were performed to determine whether the elevated Sl/Sld splenic CFU-F numbers were due to compensatory mechanisms acting in the presence of a functionally abnormal CFU-F population or to a nonspecific response to chronic anemia. Comparison of the functional ability of Sl/Sld splenic fibroblasts to produce granulocyte/macrophage colony-stimulating activity with +/+ splenic fibroblasts demonstrated that there was no difference. Similar results were obtained for Sl/Sld and +/+ femoral fibroblasts. Analysis of CFU-F in W/Wv mice revealed an approximately 3-fold higher number of splenic CFU-F than in either +/+ or heterozygous (W/+ and Wv/+) littermates. Since the anemia in W/Wv mice is attributed to a hemopoietic stem cell defect and that of the Sl/Sld mice is attributed to a microenvironmental defect, we suggest that the increased splenic CFU-F number in Sl/Sld mice is not specifically due to the microenvironmental defect, but is part of a general response to hemopoietic stress.     

Engraftment of a clonal bone marrow stromal cell line in vivo stimulates hematopoietic recovery from total body irradiation.

Whether bone marrow stromal cells of donors contribute physiologically to hematopoietic stem cell reconstitution after marrow transplantation is unknown. To determine the transplantability of nonhematopoietic marrow stromal cells, stable clonal stromal cell line (GB1/6) expressing the a isoenzyme of glucose-6-phosphate isomerase (Glu6PI-a, D-glucose-6-phosphate ketol-isomerase; EC 5.3.1.9) was derived from murine long-term bone marrow cultures and made resistant to neomycin analogue G418 by retroviral gene transfer. GB1/6 cells were fibronectin+, laminin+, and collagen-type IV+ and collagen type I-; these GB1/6 cells supported in vitro growth of hematopoietic stem cells forming colony-forming units of spleen cells (CFU-S) and of granulocytes, erythrocytes, and macrophage/megakarocytes (CFU-GEMM) in the absence of detectable growth factors interleukin 3 (multi-colony-stimulating factor), granulocyte/macrophage colony-stimulating factor, granulocyte-stimulating factor, or their poly(A)+ mRNAs. The GB1/6 cells produced macrophage colony-stimulating factor constitutively. Recipient C57BL/6J (glucose-6-phosphate isomerase b) mice that received 3-Gy total-body irradiation and 13 Gy to the right hind limb were injected i.v. with GB1/6 cells. Engrafted mice demonstrated donor-originating Glu6PI-a+ stromal cells in marrow sinuses in situ 2 mo after transplantation and a significantly enhanced hematopoietic recovery compared with control irradiated nontransplanted mice. Continuous (over numerous passages) marrow cultures derived from transplanted mice demonstrated G418-resistant, Glu6PI-a+ stromal colony-forming cells and greater cumulative production of multipotential stem cells of recipient origin compared with cultures established from irradiated, nontransplanted control mice. These data are evidence for physiological function in vivo of a transplanted bone marrow stromal cell line.     

The ultrastructure of hematopoietic stroma on cellulose ester membranes implanted intraperitoneally into Sl/Sld and Sl+/Sl+ mice

The structural features of hematopoietic stromal elements forming on cellulose ester membranes (CEM) implanted intraperitoneally into hematopoietically impaired, anemic Sl/Sld mice and their normal Sl+/Sl+ littermates were compared by combined light and electron microscopy. The generally thicker, multilayered stroma lining the Sl+/Sl+ CEM implants developed from a bed--a syncytium--of large, highly pleomorphic macrophage-type lining cells whose filopodial extensions exhibited extensive interactions (i.e., nurse cell interactions) with both stromal and hematopoietic elements. In contrast, the thinner stromal layers lining the CEM of Sl/Sld mice formed from a base of dysplastic lining elements. These CEM-lining macrophage-type cells had much reduced cytoplasmic volumes, less extensive interactive surface projections, and an absence of select types of cytoplasmic organelles (e.g., membrane-bound crystalline inclusions). These observations suggest that the reduction of cell layering and, in turn, hematopoietic support activity, is due to an impaired interactive capacity of these elemental lining cells, i.e., pleomorphic macrophagic cell types, in the hematopoietically impaired strain of Sl/Sld mice.     

Expression of a selectable gene transferred by a retroviral vector to hematopoietic stem cells and stromal cells in murine continuous bone marrow cultures

Retroviral-mediated gene transfer to multipotent and committed hematopoietic stem cells and marrow stromal cells was evaluated in long-term bone marrow cultures (LTBMCs). The retroviral vector pZIP-Neo(SV)(X) carrying the bacterial neomycin resistance (neor) gene that confers resistance to the neomycin analog G418 in mammalian cells was packaged in a Moloney envelope either as a replication-competent or replication-defective virus. Virus was introduced by infection of long-term marrow cultures at day 7. During a period of 12 weeks in culture, 10%-50% of harvested hematopoietic progenitor cells that formed differentiated CFU-GEMM colonies in response to pokeweed mitogen-containing spleen cell-conditioned medium (SCCM) and erythropoietin expressed the neor gene. In contrast, 1%-10% of hematopoietic progenitor cells that formed colonies in agar in response to WEHI-3B- or L-cell-conditioned medium expressed resistance to G418. The percentage of resistant progenitors was not detectably enhanced when replication-competent Moloney murine leukemia virus (M-MuLV) was present as helper virus, even though M-MuLV infected greater than 90% of cells in the long-term marrow cultures. In a separate CFU-F assay, 12%-17% of the adherent stromal cells in LTBMCs were found to express the neor gene. Thus gene transfer is limited by the fraction of progenitor cells that can integrate and express the transferred genetic sequences, rather than by the fraction of cells that are initially infected by the vector.     

W/Wv marrow stromal cells engraft and enhance early erythropoietic progenitors in unconditioned Sl/Sld murine recipients

Transplantation of marrow stromal cells may provide a means of modulating hematopoiesis and serve as a form of cell therapy. We employed a murine transplant model using Sl/Sl(d) mice, which have macrocytic anemia due to defective expression of stem cell factor (SCF) on bone marrow stromal cells. Donor cells were derived from the complementary mutant strain W/W(v), which also exhibit anemia, due to mutations in c-kit, the SCF receptor expressed on hematopoietic stem cells. The strength of this model is that any correction of the Sl/Sl(d) anemia from the infusion of W/W(v) stromal cells can be attributed to the effect of the stromal cells and not to contaminating W/W(v) hematopoietic stem cells, a major concern in experiments involving wild-type animals. Cultured stromal cells were infused into unconditioned non-splenectomized Sl/Sl(d) mice. Engraftment of donor stromal cells reached levels of up to 1.0% of total marrow cells 4 months post transplant. However, stromal engraftment was not detectable in the spleen. Recipients of W/W(v) stroma showed a significant increase in the committed erythroid progenitors compared with those receiving Sl/Sl(d) stromal cells: 109 +/- 26 vs 68 +/- 5 CFU-E per 10(5) BMC, P = 0.002; 25 +/- 10 vs 15 +/- 5 BFU-E per 10(5) BMC, P = 0.037, for W/W(v) and Sl/Sl(d) stroma recipients, respectively. Despite this increase in erythroid progenitors, the anemia was not corrected. Our data suggest that in this murine model, splenic erythropoiesis may influence stromal cell therapy, and that higher levels of marrow engraftment may be necessary to obtain a clinically significant effect.     

Increased longevity of hematopoiesis in continuous bone marrow cultures and adipocytogenesis in marrow stromal cells derived from Smad3(-/-) mice

OBJECTIVE: To determine the role of Smad3 in modulating hematopoiesis, continuous bone marrow cultures were established from Smad-/- mice, and the longevity of hematopoiesis and extent of adipogenesis in the supportive hematopoietic microenvironment were compared to those from cultures of control, Smad3+/+ or heterozygous Smad3+/- mice. MATERIALS AND METHODS: Long-term bone marrow cultures (LTBMCs) were established from Smad3+/+, Smad3+/-, or Smad3-/- mice. On a weekly basis, the number of cobblestone islands, number of nonadherent cells, confluence of the adherent cells, or CFU-GEMM colonies was determined. Bone marrow stromal cell lines were established and cobblestone island production on these cell lines determined in the presence of nonadherent cells from week-42 Smad3-/- or week-4 C57BL/6J LTBMCs. RESULTS: Initial proliferative capacity of the LTBMCs was similar in all groups through week 20, at which time there was an increase in cobblestone islands and production of nonadherent cells and CFU-GEMM colonies in the Smad3-/- group. By week 28, only the Smad3-/- LTBMCs had significantly maintained increased production of these parameters. Maintenance of cobblestone islands indicative of the most primitive hematopoietic progenitor cells persisted past 45 weeks in Smad3-/- cultures. The Smad3-/- stromal cell line also demonstrated increased support of cobblestone island production when incubated with nonadherent cells from week-42 Smad3-/- or week-4 C57BL/6J LTBMCs. Evaluation of adipocytogenesis in stromal cells showed significantly greater accumulation of adipocytes in lines from Smad3-/- than from Smad3+/+ mice. CONCLUSIONS: These data provide evidence for a significant effect of deletion of the Smad3 signaling pathway in increased hematopoiesis in LTBMCs and support the negative regulatory influence of TGFbeta signaling on adipocytogenesis and long-term hematopoiesis in vitro.     

Ectopic implantation studies using Sl/Sld marrow and recipients

Marrow from Sl/Sld mice (in which the hemopoietic stromal microenvironment is defective), when implanted beneath the renal capsule of a normal littermate, produces an ectopic marrow site containing the same number of stem cells (CFU-S) and nearly as many GM-CFC as that obtained by implanting marrow from a normal littermate. On the other hand, a marrow plug from an Sl/Sld donor implanted beneath the renal capsule of an Sl/Sld littermate produces less than half the number of CFU-S and about 10% of the number of GM-CFC. This suggests that the recipient of the ectopic implant can contribute in some way to the stromal environment of the grafted marrow.     

Effects in vivo of purified recombinant human activin and erythropoietin in mice.

Activin has been shown to act in vitro as an erythroid specific enhancing activity for erythropoietin (epo)-stimulated erythroid (BFU-E) and multipotential (CFU-GEMM) progenitor cells. To evaluate effects in vivo, purified recombinant activin-A and epo were administered s.c. to hypertransfused polycythemic mice for analysis of iron (59Fe) uptake, and to previously untreated mice for effects on reticulocyte release and proliferation of bone marrow (BM) and spleen (Spl) hematopoietic progenitors (CFU-GEMM, BFU-E, CFU-GM) and BM stem (CFU-S) cells. Activin alone had no effect in polycythemic BDF1 mice, but synergised with epo to significantly enhance 59Fe-incorporation into erythrocytes. In untreated C3H/HeJ mice, a single dose of activin enhanced reticulocyte release in 24 h to the level seen with epo. Activin plus epo did not further enhance reticulocyte release. Reticulocyte release was still apparent at day 4 in mice given epo twice a day for 3 days, but not in mice given activin twice a day for 3 days. Activin or epo each significantly enhanced the percent cells in S-phase of BM and Spl CFU-GEMM, BFU-E and CFU-GM in C3H/HeJ, W/Wv and Sl/Sld mice and BM CFU-S in BDF1 mice. The combination of epo plus activin did not further enhance proliferation. These results demonstrate activin's erythropoietic enhancing activities in vivo, and also activin and epo induction of enhanced proliferation of non-erythroid, as well as erythroid progenitors.     

Increased longevity of hematopoiesis in continuous bone marrow cultures derived from NOS1 (nNOS, mtNOS) homozygous recombinant negative mice correlates with radioresistance of hematopoietic and marrow stromal cells

OBJECTIVE: Neuronal nitric oxide synthase (NOS1, mitochondrial NOS, neuronal NOS) homozygous deletion recombinant negative mice demonstrate ionizing irradiation resistance in vivo, attributable to the decrease in mitochondrial-localized production of peroxynitrite, a potent lipid toxic free radical species resulting from the combination of nitric oxide and superoxide. The present studies were designed to determine whether reduced mitochondrial generation of toxic radical oxygen species in NOS1-/- mice also increased the longevity of hematopoiesis in continuous bone marrow cultures and conferred radioresistance to cells in vitro. MATERIALS AND METHODS: Long-term bone marrow cultures (LTBMCs) were established from NOS1-/- and NOS1+/+ littermate mice. Radiation resistance of hematopoietic and marrow stromal cells was measured. Cell cycle analysis and measurement of glutathione and glutathione peroxidase were carried out on irradiated clonal bone marrow stromal cell lines. RESULTS: A significant increase in longevity of hematopoiesis was detected in NOS1-/- mouse LTBMCs for over 64 weeks in culture compared to 20 weeks for NOS1+/+ mouse LTBMCs (p < 0.001). Permanent bone marrow stromal cell lines derived from NOS1-/- mouse LTBMCs demonstrated increased radioresistance in vitro reflected by an increased shoulder on the survival curve with n = 32.15 +/- 1.21 compared to NOS1+/+ cells n = 10.47 +/- 3.2 (p = 0.0026), interleukin-3-dependent NOS1-/- hematopoietic progenitor cell lines also demonstrated decreased apoptosis after 10 Gy irradiation. Both pre- and postirradiation stabilization of the cellular antioxidant pool was detected in NOS1-/- cells. NOS1-/- cells showed a prolonged G1 cell cycle arrest after 10 Gy. CONCLUSIONS: Prolonged hematopoiesis in LTBMCs correlates with intrinsic radioresistance of hematopoietic and marrow stromal cells from NOS1-/- mice. The data confirm the importance to hematopoiesis of mitochondrial localized nitric oxide in both radioresistance and longevity of hematopoiesis in continuous bone marrow cultures.     

Resistance of the stromal cell in murine long-term bone marrow cultures to damage by ionizing radiation

The ability of bone marrow stromal cells to survive and function after exposure to ionizing radiation remains controversial. Therefore, we used the murine long-term bone marrow culture system to analyze the effects of single doses of ionizing radiation (9-500 Gy) on the function of a preexisting, nearly confluent stroma that was supportive of hematopoiesis. Hematopoiesis ceased promptly in all the irradiated cultures and did not recover unless fresh marrow cells were inoculated. Radiation doses less than or equal to 100 Gy caused no obvious morphologic change in the cells. Total RNA, total protein, and collagen synthesis declined by 35%-60% within two days after even 9 Gy; but radiation doses up to 100 Gy caused minimal or no additional decline. Although RNA synthesis recovered nearly to normal within three weeks after radiation doses less than 100 Gy, total protein and collagen synthesis remained suppressed. Normal adherent layers irradiated with 9-50 Gy supported long-term hematopoiesis by fresh Sl/Sld marrow cells, although Sl/Sld marrow did not demonstrate sustained hematopoiesis when cultured in plain culture dishes or over normal stroma irradiated with 200 Gy. Thus, bone marrow stromal cells in long-term cultures did not show evidence of substantial cell death over at least the six-week period studied after irradiation with as much as 100 Gy, and they maintained hematopoietic supportive functions when irradiated with up to at least 50 Gy.     

Radiobiological properties of the human hematopoietic microenvironment: contrasting sensitivities of proliferative capacity and hematopoietic function to in vitro irradiation

We describe the effects of in vitro irradiation on the proliferative capacity and hematopoietic supportive function of human marrow stromal cells. To assess the effects on the proliferative capacity of stromal progenitors and differentiated fibroblasts, marrow cell suspensions and trypsin-dispersed marrow fibroblasts were treated with a single dose of gamma radiation at 100 rad/min. Fibroblastic progenitors (CFU-F) showed an exponential decrease in colony formation with increasing doses of irradiation, with a Do slightly higher than that of granulomonopoietic progenitors (CFU-GM); Do values for CFU-F and CFU-GM were 130 and 115, respectively. However, although the CFU-F survival curve exhibited a shoulder (n = 1.3), the CFU-GM curve did not (n = 1.0), indicating that only fibroblastic progenitors have the potential to repair irradiation- induced damage. Passaged marrow fibroblast colony-forming cells also showed a shouldered exponential survival curve with a Do of 110 and n value of 1.4. Marrow stromal progenitors giving rise to adherent layers in long-term marrow cultures also demonstrated a highly radiosensitive proliferative capacity. Stromal layers derived from irradiated marrow suspensions failed to establish adherent layers after relatively low doses of irradiation (over 240 rad) in a dose-response manner. To assess any functional damage in stromal progenitors surviving irradiation, stromal layers derived from marrow suspensions irradiated up to 240 rad were cocultured with freshly isolated autologous hematopoietic cells and assayed for their capacity to support prolonged CFU-GM production. Confluent stromal layers derived from irradiated marrow suspensions sustained CFU-GM production as well as controls. To study the effects of irradiation on the hematopoietic supportive capacity of established marrow-derived stromal layers, 4 to 6-week-old adherent layers were irradiated as described and cocultured with autologous marrow cells enriched for colony-forming cells. Stromal layers irradiated up to 1,320 rad sustained prolonged CFU-GM production, indicating that the hematopoietic supportive function remained intact at this dose of irradiation. In conclusion, we demonstrated that the proliferative capacity of human marrow stromal progenitors, as well as that of their differentiated descendants, is quite sensitive to in vitro radiation, while the hematopoietic supportive function of differentiated stromal cells is relatively resistant to the effects of radiation.     

Phorbol 12-myristate 13-acetate-induced development of functionally active mast cells in W/Wv but not Sl/Sld genetically mast cell-deficient mice

The normal skin and other tissues of adult genetically mast cell-deficient WBB6F1-W/Wv or WCB6F1-Sl/Sld mice contain less than 1.0% the number of mast cells present in the corresponding tissues of the congenic normal (+/+) mice. We previously reported that mature dermal mast cells developed locally in the skin of W/Wv, but not Sl/Sld, mice at sites of chronic idiopathic dermatitis. We now report that the repeated application of phorbol 12-myristate 13-acetate (PMA) to the ear skin of either W/Wv or +/+ mice induces both dermatitis and a striking and dose-dependent increase in the number of dermal mast cells. The number of dermal mast cells at sites treated for 6 weeks with 5 micrograms PMA, three times per week, was 39 +/- 7/mm2 and 305 +/- 34/mm2 for W/Wv and +/+ mice, respectively; the corresponding values for vehicle-treated skin were 1.5 +/- 1.0/mm2 and 145 +/- 8/mm2, respectively. The PMA-induced dermal mast cells in W/Wv mice appeared mature by morphology, stained with the heparin-binding fluorescent dye, berberine sulfate, and were competent to express IgE-dependent passive cutaneous anaphylaxis responses. The development of mast cells was a local, not systemic, effect of PMA treatment. PMA treatment also induced dermatitis in both WCB6F1-Sl/Sld and +/+ mice, but was associated with increased numbers of dermal mast cells only in the WCB6F1(-)+/+ mice. PMA treatment had no detectable effect on the ability of bone marrow-derived cultured mast cells to survive in the skin of Sl/Sld mice. These findings establish a convenient model system for analyzing factors associated with the development of endogenous populations of mast cells in genetically mast cell-deficient W/Wv mice.     

Effects of low dose rate irradiation on human marrow hematopoietic and microenvironmental cells: sparing effect upon survival of stromal and leukemic cells

The effects of different dose rates of in vitro irradiation on the proliferative capacity of marrow stromal, hematopoietic and leukemic colony-forming cells (CFC) are described. Marrow cell suspensions, HL-60 cells and trypsin-dispersed fibroblasts were irradiated at 5 or 45 cGy/min and then assayed for CFC. Irradiation at low (5 cGy/min) compared to high (45 cGy/min) dose rate showed a significant difference in survival of stromal and of HL-60 cells, but not of hematopoietic progenitors: the respective D0 values were 170 and 120 (p = 0.003) for marrow fibroblastic progenitors (CFU-F); 145 and 110 (p = 0.005) for passaged marrow fibroblasts (CFU-F); 170 and 140 (p = 0.045) for HL-60 cells; 85 and 85 for multipotential CFC (CFU-mix); 125 and 120 for erythroid progenitors (BFU-E); and 115 and 120 for granulomonopoietic progenitors (CFU-GM) (p = 0.5 for hematopoietic clonogenic cells). Marrow suspensions did not establish confluent stromal layers in long-term marrow cultures following irradiation with 600 cGy at 45 cGy/min, whereas after 840 cGy at 5 cGy/min confluent stromal layers were obtained. This indicates that low dose rate-sparing effect applies to all stromal cell progenitors. Confluent stromal layers derived from progenitors surviving irradiation sustained hematopoiesis as well as controls when co-cultured with fresh hematopoietic cells. Adherent layers in long-term marrow cultures irradiated after establishment with doses less than or equal to 1500 cGy at 5 or 45 cGy/min also showed normal hematopoietic supportive function when co-cultured with freshly isolated hematopoietic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Biologic effects of in vitro X-irradiation of murine long-term bone marrow cultures on the production of granulocyte-macrophage colony-stimulating factors

The production of colony-stimulating factor (CSF) by murine long-term bone marrow culture (LTBMC) was studied by a technique involving measurement of colony formation in agar overlay by fresh marrow target cells. Colonies were removed and microscopically examined for morphology and histochemistry. LTBMCs were exposed to x-irradiation at 200 rad/min prior to the overlay. Nonirradiated control LTBMCs induced 51.5 +/- 11 granulocyte-macrophage colonies per 2 X 10(5) target cells. Irradiation of LTBMCs to 6000 rad revealed a six-fold plateau-maximum increase in the number of colonies. There was occasional appearance of macroscopic mixed colonies containing granulocytes, macrophages, and megakaryocytes over irradiated but not control LTBMCs. Irradiated cells in the adherent stromal layer of LTBMCs continuously produced CSF that was detectable in the cell-free supernatants for up to seven weeks after irradiation and after doses as high as 10,000 rad. Shielding of the x-ray beam over half of the culture surface by a 10-half-value-layer lead block produced increased colony formation by target cells near the exposed surface area. The data indicate that CSF production by adherent cells within LTBMC persists after supralethal doses of x-irradiation. The mechanism of the increased colony formation by target cells overlaid on irradiated stromal cells involves factors relative to the local microenvironment.     

The cause of anemia in mutant mice of Sl/Slt genotype: hypoplasia in bone marrow and restricted hyperplasia in spleen

The cause of the severe anemia in Sl/Sld mice is attributed to (1) hypoproduction of erythrocytes due to a defect in the erythropoietic microenvironment and (2) bleeding from stomach ulcers. Sl/Slt mice also showed a moderate anemia, but bleeding from stomach ulcers was excluded as a cause of the anemia, because no significant amount of radioactivity was excreted in feces after the injection of 59Fe-labeled erythrocytes. The activity of erythropoiesis in the bone marrow and spleen was compared between Sl/Slt and congenic +/+ mice using three different criteria: the number of erythroblasts, 59Fe incorporation, and the number of erythropoietic precursor cells. All three parameters in the femur were lower, and those in the spleen were higher in Sl/Slt mice than in +/+ mice, suggesting that the low erythropoietic potential in the bone marrow of Sl/Slt mice is partially compensated by the spleen. In fact, splenectomy aggravated the anemia of Sl/Slt mice. The enhanced erythropoiesis in Sl/Slt spleens may explain our previous finding that numbers and sizes of spleen colonies were normal when bone marrow cells were injected into irradiated Sl/Slt mice. Sl/Slt mice may be a useful model for studying biological characteristics of the hematopoietic microenvironment.     

Infection of hematopoietic and stromal cells in human continuous bone marrow cultures by a retroviral vector containing the neomycin resistance gene

Stability and expression of the bacterial neomycin resistance gene (neor) transferred to human continuous marrow cultures by a retroviral vector [pZIP-NeoSV(X)] was evaluated over 4 weeks. Following infection of long-term human marrow cultures with pZIP-NeoSV(X), 10-15% of the stromal cells demonstrated high replating efficiency in a dose of the neomycin analogue G418 that was toxic to stromal cells from uninfected cultures. In contrast, G418 resistance was detected in less than or equal to 1% of GM-CFUc and CFU-GEMM derived from the same virus-infected compared to control cultures. Infection of human CFU-GEMM enriched 100 X by monoclonal antibody selection with pZIP-NeoSV(X) did not increase the percentage of neor progenitors. Marrow cells from cultures infected with pZIP-NeoSV(X) and a replication competent amphotropic virus transferred the vector and G418 resistance to HeLa cells at a frequency of 1/10(5) for nonadherent and 1/10(4) for adherent cells. Two established human hematopoietic (HL60 and K562) and one stromal cell line (KM101) stably expressed the neor gene. Thus, a higher efficiency of infection and expression of a gene transferred by pZIP-NeoSV(X) to permanent human hematopoietic tumor cell lines and fresh marrow stromal cells contrasts with a lower level of expression in fresh CSF-dependent human hematopoietic stem cells.     

Hematopoietic stem cell- and marrow stromal cell-specific requirements for gamma irradiation leukemogenesis in vitro

The hematopoietic and stromal cell-specific properties of the cells involved in gamma irradiation leukemogenesis in vitro were defined. Cocultivation of clonal factor-dependent (FD), interleukin 3 (IL-3)-dependent cell lines 32D cl 3 or B6SUtA, or dual IL-3-/granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell lines FDC-P1 or bg/bg d64 was carried out with clonal stromal cell lines D2XRII, GB1/6, +/+ 2.4, or Sld3. FD cell lines were added to control or 5000-cGy-irradiated plateau phase monolayer cultures of each stromal cell line, and parameters of stem cell engraftment and malignant transformation in vitro were quantitated. Cobblestone island formation by FD cells, cumulative production of nonadherent hematopoietic cells, and evolution of tumorigenic factor-independent (FI) subclonal lines were quantitated over 5-8 weeks. There was no detectable evolution of FI sublines with 32D cl 3, B6SUtA, or bg/bg d64 cells cocultivated with control or irradiated Sld3 stromal cells. IL-3-dependent cell lines 32D cl 3 or B6SUtA formed small 10- to 49-cell cobblestone "clusters" at low frequency on control or irradiated D2XRII, showed limited proliferation for less than 1 week, and showed no detectable evolution of FI cell lines. Subclones of 32D cl 3 derived by transfection and expression of recombinant oncogenes v-sis, or c-myc, or the epidermal growth factor receptor remained factor dependent and did not transform to factor independence after cocultivation with irradiated stromal cell lines. In contrast, cell line bg/bg d64, and each of seven subclonal lines of FDC-P1, including subclones selected for growth in GM-CSF, formed abundant cobblestone island colonies of greater than or equal to 50 cells on irradiated D2XRII stromal cells, produced non-adherent cells over 5-8 weeks, and showed evolution of tumorigenic FI subclonal lines. The data provide evidence for stable biological differences in both the hematopoietic and stromal cell components of the in vitro model of gamma irradiation leukemogenesis.     

Determinants and characteristics of mean corpuscular volume and hemoglobin concentration in white HFE C282Y homozygotes in the hemochromatosis and iron overload screening study

Elevated mean corpuscular volume (MCV) is common in persons with hemochromatosis associated with HFE C282Y homozygosity. We evaluated data from the subset of non-Hispanic white participants in the Hemochromatosis and Iron Overload Screening Study to determine if elevated MCV in C282Y homozygotes is related to this genotype or to serum iron measures. Regression analysis was used to model MCV and Hb from transferrin saturation (TfSat), serum ferritin (SF), mean corpuscular hemoglobin concentration, red blood cell count, age, HFE genotype, Field Center, and presence of liver-related abnormalities in C282Y homozygotes and control subjects without HFE mutations (wt/wt genotype). Mean MCV was higher in C282Y homozygotes than in HFE wt/wt controls (94.4 vs. 89.7 fL in women; 95.3 vs. 91.2 fL in men; P < 0.0001 for both). These differences were largely associated with increased mean TfSat and SF in C282Y homozygotes. Adjusted mean MCV was 92.0 fL (95% confidence interval, 91.1, 92.9) in female C282Y homozygotes and 90.9 fL (90.3, 91.5) in controls. Among women with SF in the reference range 20-200 microg/L, adjusted mean MCV was 92.9 fL, (91.7, 94.2) in C282Y homozygotes, 1.8 fL higher than in controls (P = 0.013). The adjusted mean MCV of male C282Y homozygotes and controls was similar (P = 0.30). Adjusted mean Hb was 0.2 g/dL higher in women with C282Y/C282Y than in controls. Greater mean MCV in C282Y homozygosity reflects increased mean TfSat and mean SF in men and women; an additional effect of genotype on MCV and Hb was detected in women.