In vivo effect of murine recombinant interleukin 3 on early hemopoietic progenitors

Normal and irradiated mice were perfused with recombinant interleukin 3 (rIL3) and the number of early hemopoietic progenitors (CFU-S) was quantified in different organs. Normal mice perfused with rIL3 for 3 or 7 days showed a dramatic increase in the number of CFU-S in the spleen, liver and blood, while the bone marrow CFU-S number was slightly decreased. The total number of CFU-S per animal was only slightly increased; the major effect of rIL3 perfusion was thus on the distribution of CFU-S in the mice. Lethally irradiated mice injected with 5 X 10(4) syngeneic bone marrow cells and perfused with rIL3 were killed after 10 days. A significant increase in both the number and size of the splenic colonies was observed. The CFU-S content of these colonies, determined in a second set of irradiated mice, was increased by more than 25-fold, indicating that in conditions of hemopoietic depletion, rIL3 vastly increased the self-renewal capacity of early progenitors.     

Inhibition of resistance to hemopoietic allo-grafts in granulocyte colony-stimulating factor transgenic mice

Transplanted allogeneic marrow cells often fail to engraft in a lethally irradiated host. This phenomenon, termed resistance to allogeneic marrow grafts or allo-resistance, is well documented, although its mechanism is not yet understood. Transplantation of major histocompatibility complex disparate allogeneic marrow cells into mice transgenic for granulocyte colony-stimulating factor (G-CSF) showed donor-derived spleen colonies (CFU-S) and resulted in stable allogeneic chimerism with excellent survival (100% up to 40 days and 89% up to 120 days). Under the same experimental conditions, all the littermate controls failed to show CFU-S and died shortly after marrow transplantation. Thus, resistance to allogeneic marrow cells appeared to be severely impaired in this transgenic mouse. The observation that neutralizing antibody against G-CSF restored allo-resistance in G-CSF transgenic mice and that CFU-S was inducible upon administration of recombinant G-CSF using a mini-osmotic pump in non-transgenic recipients, suggests that an elevated level of this cytokine is important for the inhibition of allo-resistance. Thus, G-CSF was found to play a role in allogeneic resistance to marrow grafts and the G-CSF-transgenic mice provide a useful model to study the inhibition of the resistance. The inhibition of allo-resistance may be useful in preparing allogeneic bone marrow chimeras in both experimental and clinical settings.     

Effect of pregnancy on the activity of hematopoietic stem cells

Effect of allogenic and syngenic pregnancy on the activity of multifunctional hematopoietic stem cells of bone marrow and spleen was studied. It was observed that bone marrow of mice mated syngenically and allogenically was more capable of restoring hematopoiesis in lethally irradiated recipients as compared to the bone marrow of nonpregnant mice. Increased number of colony forming hematopoietic cells (CFU-S) was recorded also in the spleen of female mice with syngenic pregnancy. When the spleen of pregnant mice mated allogenically, was the source of the multifunctional hematopoietic spleen cells, the animals displayed suppressed capability to restore hematopoiesis which manifested by a decreased number of CFU-S. Splenectomy appeared to have no influence upon the number of CFU-S in the bone marrow of pregnant mice mated allogenically.     

c-kit<low Pluripotent hemopoietic stem cells form CFU-S on day 16

Using Ly5 congenic mice, we characterized the early differentiation step of pluripotent hemopoietic stem cells. Lineage- (Lin-)/CD71- cells in the bone marrow cells were separated into major histocompatibility complex (MHC) class I(high)/c-kit(low) and MHC class I(high)/c-kit相似文献   

Human Fetal Liver Cells Induce Colonies in Spleen of Lethally Irradiated Mice

Murine hematopoietic tissues contain cells which, upon injection into lethally irradiated mice, produce nodules on the surface of their spleen (colony-forming unit—spleen; CFU-S). The exact hierarchical level of the hematopoietic progenitors which give rise to CFU-S is not fully established; however, cell populations highly enriched for repopulating stem cells appear to contain a high percentage of CFU-S. The experiments reported here involved the injection of human fetal liver cells into mice, under conditions similar to those of the CFU-S test. These data demonstrate that human fetal liver cells are able to induce spleen colonies (tentatively called human CFU-S) when injected into lethally irradiated mice. The number of CFU-S was increased by prior purification of human fetal liver cells. When mice were injected with human fetal liver cells inactivated by irradiation, no human CFU-S were observed. Positive staining of cells found in spleen colonies, using monoclonal antibodies specific for various human determinants, indicated the human origin of part of them. The presence of human cells within the colonies was further confirmed by in situ hybridization using a probe specific for human DNA. A mean of 30–40% of analyzed colonies was thus shown to contain some patches of human cells. These data confirm that human hematopoietic cells are able to seed, proliferate, and differentiate in a murine microenvironment.     

A new assay method for late CFU-S formation and long-term reconstituting activity using a small number of pluripotent hemopoietic stem cells

We have previously reported that Lin-/CD71-/MHC class Ihigh/c-kit相似文献   

Local environmental factors determine hematopoietic differentiation of neural stem cells

Stem cells exhibit unique properties and hold high therapeutic promise, but factors influencing their differentiation after transplantation need to be recognized and defined for this promise to be fully met. Here, we demonstrate that endogenous colony-forming unit spleen (CFU-S) colonies are not generated in lethally irradiated mice transplanted with neural stem cells obtained from brain tissue of syngeneic donors. We investigated the proportion of transplanted neural stem cells that contributed to hematopoietic reconstitution and compared the distribution of transplanted cells in nonsplenectomized to that of splenectomized mice following sublethal whole-body irradiation. We also used clonogenic assays, colony assays, and histochemical analyses to explore conditions under which transplanted, beta-galactosidase-tagged neural stem cells underwent hematopoietic differentiation. Our results suggest that neural stem cells do undergo extramedullary hematopoiesis, even while no endogenous hematopoietic colonies develop in the spleen. Furthermore, we found that neural stem cells effectively colonized the bone marrow of splectomized recipients. We conclude that the hematopoietic differentiation of neural stem cells is highly dependent on the extramedullary environment. We also conclude that the bone marrow does not provide an environment supportive of hematopoietic differentiation by neural stem cells.     

Accelerated recovery from gamma-irradiation-induced leukopenia in mice by the biscoclaurine alkaloid, Cepharanthin--comparison with recombinant human granulocyte colony-stimulating factor.

The effect of a biscoclaurine alkaloid drug, Cepharanthin (CE), on recovery from severe leukopenia induced by whole body gamma-irradiation at a dose of 3 Gy, was compared with that of recombinant human granulocyte colony-stimulating factor (rhG-CSF). Daily intraperitoneal administration of 100 micrograms CE into the irradiated mice significantly prevented decrease of leukocyte numbers in the peripheral blood and accelerated recovery from leukopenia to normal. The activity of CE was somewhat weaker than that of 20,000 units of rhG-CSF when administered daily subcutaneously. In cell composition of peripheral leukocytes, CE increased the numbers of polymorphonuclear (PMN) cells and lymphocytes compared with those of the irradiated controls. Stronger change in PMN cell numbers than with CE was induced by administration of rhG-CSF. No significant increment of the activity to form colonies in spleen (colony-forming unit in spleen; CFU-S) was observed in bone marrow cells from irradiated donor mice administered CE, though CE induced CSF production into sera. Administration of rhG-CSF stimulated the CFU-S activity. These results indicate that CE increased recruitment of PMN cells from the marginal pool rather than the stimulation of pluripotent stem cells in the bone marrow.     

Immune responsiveness of irradiated mice reconstituted with complement receptor positive CLR+ or negative CLR--lymphocytes from spleen or bone marrow.

Spleen and bone marrow cells depleted of CRL cannot restore immune reactivity of irradiated mice to sheep red blood cells SRBC. The response to anti-SIII, a T-independent antigen does not change when irradiated animals are repopulated with spleen cells or spleen cells depleted of CRL. Spleen cells of 6-day-old mice having low number of CRL, in contrast to spleen cells of adult animals, could not reconstitute irradiated recipients.     

蝎毒多肽对辐射损伤小鼠骨髓造血干细胞及祖细胞的作用

目的 探讨不同蝎毒多肽(scorpion venom peptide,SVP)组分对辐射后机体造血干细胞及祖细胞恢复的作用.方法 6.0 Gy X射线一次性全身照射,制作辐射损伤小鼠模型.内源性脾结节法观察照射后第10天脾集落形成单位(CFU-S)的变化.用甲基纤维素半固体培养基培养骨髓混合集落生成单位(CFU-Mix),观察体内外给药方法及照射后不同时间对CFU-Mix生成的影响.结果 (1)体内实验:SVPⅣ组分处理后的CFU-S数明显高于照射对照组(P＜0.05);SVPV组分CFU-S数量与照射对照组差异无统计学意义.照射后各SVP组CFU-Mix的数量均高于照射对照组,差异有统计学意义(P＜0.05).(2)体外实验:与照射对照组相比,体外分别单独加入SVPⅣ、Ⅴ组分以及细胞因子(IL-6和SCF)均能够促进CFU-Mix的增殖;而SVPⅣ、Ⅴ组分分别与细胞因子联合应用对CFU-Mix生成的促进作用更为明显,其中Ⅳ组分效果更强,与照射对照组相比差异均有统计学意义(P＜0.05).结论 SVP具有保护辐射损伤小鼠造血干细胞及祖细胞,加速其增殖能力恢复的作用.     

Hemopoietic repopulating potential of subcutaneous exudate cells

Inflammatory exudate (SE) cells were collected from subcutaneous coverslips in mice and transferred into lethally irradiated (1,000 r) recipients. Eight days after transplantation ⁵⁹Fe incorporation in the spleen and bone marrow was significantly greater than in controls treated with the suspending medium only. One hundred percent of mitoses were of the T6T6 karyotype in the marrow and spleen when SE cells were obtained from CBA/T6T6 donors. The repopulating potential of SE cells, however, lagged significantly, behind that of bone marrow cells and the failure to observe consistently macroscopic spleen colonies calls into question whether the observed regeneration was due to pluripo-tent stem cells. Radioautographic studies with ³H-TdR showed that the majority of SE cells had recently been generated, but long-lived, noncycling cells of lymphoid and monocytoid morphology were also present in the exudate.     

Modification of macrophage differentiation: dimethylnitrosamine induced alteration in the responses towards the regulatory signals controlling myelopoiesis

Results from our laboratory have demonstrated that the alteration in cellular immunity (CMI) resulting from exposure to dimethylnitrosamine (DMN) in vivo is due to changes in myelopoiesis. Bone marrow stem cells showed no alterations in their capacity to generate CFU-S (pleuripotent stem cells) nor were there any changes in the number of CFU-Mix colonies (IL-3 responsive stem cells) arising from the bone marrow of DMN exposed mice. However, the generation of G/M-CSF and CSF-1 responsive colonies (CFU-G/M and CFU-M) were altered, resulting in an increase in the number of colonies. G/M-CSF colonies generated from the bone marrow stem cells obtained from DMN exposed mice also had increased numbers of cells produced by each colony (total cells/CFU). Indirect immunofluorescence studies demonstrated no changes in the granulocyte/macrophage subsets following G/M-CSF stimulation of bone marrow stem cells obtained from DMN exposed mice. However, there was no change in the total number of cells generated by CSF-1 from the marrows of DMN exposed mice as compared to vehicle treated mice. Marrow cells from DMN exposed mice cultured in vitro with G/M-CSF showed both a shift in their peak proliferative response from 48-72 h to 30-60 h and an increased proliferative response. These same marrow cells showed no shift in their kinetics but a decrease in their proliferative response to CSF-1. Examination of the sera from DMN exposed mice for alterations in the regulatory factors controlling myelopoiesis demonstrated a net decrease of CSF-1 activity but no changes in the concentrations of two inhibitory factors, transferrin and lactoferrin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The recovery of the B-cell population in adult thymectomized, lethally irradiated and bone marrow-reconstituted mice.

The recovery of the B-cell population in adult thymectomized, irradiated and bone marrow-reconstituted mice (T X BM mice was estimated at various times after bone marrow transplantation. The spleen cells to be tested were mixed with dexamethasone-resistant thymocytes (DRT) and sheep red blood cells (SRBC) and transferrred to irradiated recipients. The number of plaque-forming cells (PFC) in the spleen of the recipients was determined 7 days later. Using this functional B-cell assay a sequential appearance of the precursors of IgM-, IgG- and IgA-PFC in the spleen of T X BM mice was observed. The precursors of IgM-PFG (IgM-B cells) were present immediately after transplantation. The first IgG-B cells could be detected at 13-16 days after transplantation and the IgA-B cells finally appeared at 22 days after transplantation. The number of B cells reached a constant and normal level at 30 days after transplantation. The IgM-, IgG- and IgA-B cell development in sham-thymectomized, irradiated and bone narrow-reconstituted mice (ST X BM mice) was virtually the same as in T X BM mice.     

Evaluation of hematopoietic potential generated by transplantation of muscle-derived stem cells in mice

Muscle tissue of adult mice has been shown to contain stem cells with hematopoietic repopulation ability in vivo. To determine the functional characteristics of stem cells giving rise to this hematopoietic activity, we have performed hematopoietic reconstitution experiments by the use of muscle versus marrow transplantation in lethally irradiated mice and followed the fate of transplanted cells by Y-chimerism using PCR and fluorescence in situ hybridization (FISH) analysis. We report here that transplantation of murine muscle generate a major hematopoietic chimerism at the level of CFU-C, CFU-S, and terminally-differentiated cells in three generations of lethally irradiated mice followed up to 1 year after transplantation. This potential is totally abolished when muscle grafts were performed by the use of muscle from previously irradiated mice. As compared to marrow transplantation, muscle transplants were able to generate similar potencies to give rise to myeloid, T, B, and natural killer (NK) cells. Interestingly, marrow stem cells that have been generated in primary and then in secondary recipients were able to contribute efficiently to myofibers in the muscle tissue of tertiary recipients. Altogether, our data demonstrate that muscle-derived stem cells present a major hematopoietic repopulating ability with evidence of self-replication in vivo. They are radiation-sensitive and similar to marrow-derived stem cells in terms of their ability to generate multilineage hematopoiesis. Finally, our data demonstrate that muscle-derived hematopoietic stem cells do not lose their ability to contribute to myofiber generation after at least two rounds of serial transplantation, suggesting a potential that is probably equivalent to that generated by marrow transplantation.     

Migration of bone marrow lymphocytes in mice: immunofluorescent staining using antiallotype serum

Bone marrow cells from CSW (Igha) mice were injected intravenously into the congeneic CWB (Ighb) mice. The lymphoid tissues of the recipient CWB mice were examined for donor type surface immunoglobulin (SIg)-positive cells using fluorescein isothiocyanate-conjugated antiallotype serum Ighb anti-Igha. Donor type SIg-positive cells were rarely found in the recipients immediately after injection. However, the donor type B cells increased in the recipient's spleen from days 1-2 and reached a plateau thereafter. The present method provides a useful means for studying the migration patterns and differentiation of bone marrow lymphocytes.     

Intra-bone marrow injection of donor bone marrow cells suspended in collagen gel retains injected cells in bone marrow, resulting in rapid hemopoietic recovery in mice

We have recently developed an innovative bone marrow transplantation (BMT) method, intra-bone marrow (IBM)-BMT, in which donor bone marrow cells (BMCs) are injected directly into the recipient bone marrow (BM), resulting in the rapid recovery of donor hemopoiesis and permitting a reduction in radiation doses as a pretreatment for BMT. However, even with this IBM injection, some of the injected BMCs were found to enter into circulation. Therefore, we attempted to modify the method to allow the efficient retention of injected BMCs in the donor BM. The BMCs of enhanced green fluorescent protein transgenic mice (C57BL/6 background) were suspended in collagen gel (CG) or phosphate-buffered saline (PBS), and these cells were then injected into the BM of irradiated C57BL/6 mice. The numbers of retained donor cells in the injected BM, the day 12 colony-forming units of spleen (CFU-S) counts, and the reconstitution of donor cells after IBM-BMT were compared between the CG and PBS groups. The number of transplanted cells detected in the injected BM in the CG group was significantly higher than that in the PBS group. We next carried out CFU-S assays. The spleens of mice in the CG group showed heavier spleen weight and considerably higher CFU-S counts than in the PBS group. Excellent reconstitution of donor hemopoietic cells in the CG group was observed in the long term (>100 days). These results suggest that the IBM injection of BMCs suspended in CG is superior to the injection of BMCs suspended in PBS.     

Development of Natural Killer Cell Activity and Genetic Resistance to Bone Marrow Transplantation with Age: Effect of Neonatal Thymectomy

The effect of neonatal thymectomy on the development of splenic and bone marrow natural cell-mediated cytotoxicity and on genetic resistance to bone marrow transplantation was examined in mice. Natural cytotoxicity was measured by a ⁵¹Cr release assay; the ability to engraft foreign bone marrow was assayed by the spleen colony method. The natural cytolytic response of spleen cells increased progressively from youth to early adulthood, whereas that of the bone marrow declined during the same age period. Neonatal thymectomy significantly elevated the natural killer cell response of young mice only (4 weeks, spleen; 6 weeks, bone marrow). In other experiments, neonatally thymectomized and sham-operated mice were lethally irradiated at 4 or 6 weeks of age and injected with 2.5, 5.0 or 10 million rat marrow cells. Six days later spleen colonies were markedly reduced in both 4- and 6-week-old neonatally thymectomized mice with all rat marrow cell doses tested. Neonatal thymectomy did not alter the percentage of erythroid versus other colonies at either 4 or 6 weeks. In both thymectomized and sham-operated mice the number of colonies increased with increases in marrow cell dose. The data are suggestive of a production and dissemination to the spleen of cells involved in the natural cytotoxic response from the bone marrow.     

Recombinant adeno-associated virus-based vectors provide short-term rather than long-term transduction of primitive hematopoietic stem cells

Bone marrow stem cells collected from B6-Gpi-1a mice pretreated with 5-fluorouracil were incubated for 2 h at 37 degrees C in the presence of the recombinant adenovirus-associated virus-based vector (rAAV) SSV9. As measured in vitro immediately following transduction, SSV9 was found to be effective in transducing the primitive cobble-stone-area-forming cell (CAFC)-35 subset (60% transduction efficiency). However, this did not predict long-term expression as the presence of the transgene could not be detected six months after transplantation of 1-2 x 106 transduced bone marrow stem cells into lethally irradiated recipients. CAFC analysis of bone marrow cells and Southern blot analysis of bone marrow and spleen cells were negative, and polymerase chain reaction analysis showed less than 0.1% transduction in bone marrow cells. Therefore, based on our study we conclude that rAAV transiently transduces hematopoietic stem cells but fails to integrate into the genome, leading to the loss of the reporter gene within the first six months after transplantation in vivo.     

Administration of granulocyte colony-stimulating factor to recipients followed by intra-bone marrow-bone marrow transplantation accelerates acceptance of allogeneic bone marrow cells in mice

We have recently established a novel method for bone marrow transplantation: intra-bone marrow–bone marrow transplantation (IBM–BMT), by which the rapid recovery of donor-derived hematopoiesis can be expected even when reduced radiation doses are used. In this paper, we examine, using mice, whether the combination of pretreatment of recipients with granulocyte-colony-stimulating factor (G-CSF) and IBM–BMT can induce a more rapid recovery of donor-derived hematopoiesis than IBM–BMT alone.

We first pretreated recipients with recombinant human (rh) G-CSF (250 μg/kg/day) for 5 consecutive days (days −6 to −2). On day −1, the recipients were irradiated, and IBM–BMT was carried out on day 0. On day 12, we performed colony-forming units of spleen (CFU-S) assays. The combination of G-CSF pretreatment and IBM–BMT augmented the CFU-S counts, the weight of spleens, and the numbers of donor-derived hematopoietic cells. We next analyzed the mechanisms underlying these effects of G-CSF and found that (i) G-CSF induces Th2 polarization, which can prevent graft rejection, and (ii) G-CSF augments natural suppressor activity, which suppresses graft rejection. The combination of G-CSF pretreatment and IBM–BMT can produce the rapid recovery of donor-derived hematopoiesis and suppress graft rejection. This method would lighten the burden on patients in allogeneic BMT.