Leukocytosis in mice following long-term reconstitution with genetically-modified bone marrow cells constitutively expressing interleukin 1α or interleukin 6

Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population.

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Bisecting GlcNAc structure is implicated in suppression of stroma-dependent haemopoiesis in transgenic mice expressing N-acetylglucosaminyltransferase III

Biochemical Journal ◽

10.1042/bj3310733 ◽

1998 ◽

Vol 331 (3) ◽

pp. 733-742 ◽

Cited By ~ 8

Author(s):

Masafumi YOSHIMURA ◽

Yoshito IHARA ◽

Tetsuo NISHIURA ◽

Yu OKAJIMA ◽

Megumu OGAWA ◽

...

Keyword(s):

Bone Marrow ◽

Transgenic Mice ◽

Stromal Cells ◽

Bone Marrow Cells ◽

Adherent Cell ◽

Wild Type ◽

Spleen Cells ◽

Bisecting Glcnac ◽

Marrow Cells

Several sugar structures have been reported to be necessary for haemopoiesis. We analysed the haematological phenotypes of transgenic mice expressing β-1,4 N-acetylglucosaminyltransferase III (GnT-III), which forms bisecting N-acetylglucosamine on asparagine-linked oligosaccharides. In the transgenic mice, the GnT-III activity was elevated in bone marrow, spleen and peripheral blood and in isolated mononuclear cells from these tissues, whereas no activity was found in these tissues of wild-type mice. Stromal cells after long-term cultures of transgenic-derived bone marrow and spleen cells also showed elevated GnT-III activity, compared with an undetectable activity in wild-type stromal cells. As judged by HPLC analysis, lectin blotting and lectin cytotoxicity assay, bisecting GlcNAc residues were increased on both blood cells and stromal cells from bone marrow and spleen in transgenic mice. The transgenic mice displayed spleen atrophy, hypocellular bone marrow and pancytopenia. Bone marrow cells and spleen cells from transgenic mice produced fewer haemopoietic colonies. After lethal irradiation followed by bone marrow transplantation, transgenic recipient mice showed pancytopenia compared with wild-type recipient mice. Bone marrow cells from transgenic donors gave haematological reconstitution at the same level as wild-type donor cells. In addition, non-adherent cell production was decreased in long-term bone marrow cell cultures of transgenic mice. Collectively these results indicate that the stroma-supported haemopoiesis is compromised in transgenic mice expressing GnT-III, providing the first demonstration that the N-glycans have some significant roles in stroma-dependent haemopoiesis.

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Interleukin-6 enhances murine megakaryocytopoiesis in serum-free culture

Blood ◽

10.1182/blood.v75.12.2286.bloodjournal75122286 ◽

1990 ◽

Vol 75 (12) ◽

pp. 2286-2291 ◽

Cited By ~ 1

Author(s):

K Koike ◽

T Nakahata ◽

T Kubo ◽

T Kikuchi ◽

M Takagi ◽

...

Keyword(s):

Bone Marrow ◽

Interleukin 6 ◽

Bone Marrow Cells ◽

Early Stage ◽

Colony Growth ◽

Colony Stimulating Factor ◽

Serum Free ◽

Macrophage Colony ◽

Stimulating Factor ◽

Marrow Cells

We investigated the effect of interleukin-6 (IL-6) on murine megakaryocytopoiesis in a serum-free culture system. The addition of IL- 6 to a culture containing interleukin-3 (IL-3) resulted in a significant increase in the number of megakaryocyte colonies by bone marrow cells of normal mice. The megakaryocytic progenitors that survive exposure to 5-fluorouracil (5-FU) exhibited a more significant response to IL-6 and IL-3. Polyclonal anti-IL-6 antibody neutralized the stimulatory effect of IL-6 on megakaryocyte colony growth supported by IL-3. Delayed addition experiments and replating experiments of blast cell colonies showed that megakaryocytic progenitors are supported by IL-3 in the early stage of the development but require IL- 6 for their subsequent proliferation and differentiation. In addition, IL-6 increased the size of megakaryocytes in granulocyte-macrophage- megakaryocyte colonies. The combination of granulocyte colony- stimulating factor or granulocyte-macrophage colony stimulating factor with IL-3 resulted in an increase in the granulocyte-macrophage colony growth of bone marrow cells of 5-FU-treated mice or normal mice, respectively, but had little effect on the enhancement of pure and mixed megakaryocyte colony growth. These results suggest that IL-6 plays an important role in murine megakaryocytopoiesis.

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Phenotype and functional identity of GM-CSF–independent dendritic cells generated by long-term propagation of DC progenitor cells in bone marrow cells and skin langerhans cells

Transplantation Proceedings ◽

10.1016/j.transproceed.2005.02.017 ◽

2005 ◽

Vol 37 (1) ◽

pp. 17-19 ◽

Cited By ~ 4

Author(s):

H. Katayama ◽

Y. Hattori ◽

K. Ogata ◽

H. Yan ◽

E. Satoh ◽

...

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Progenitor Cells ◽

Langerhans Cells ◽

Bone Marrow Cells ◽

Functional Identity ◽

Gm Csf ◽

Marrow Cells

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22. Tumor Homing of Genetically Modified Bone Marrow Cells

Molecular Therapy ◽

10.1016/j.ymthe.2004.05.045 ◽

2004 ◽

Vol 9 ◽

pp. S9

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Genetically Modified ◽

Tumor Homing ◽

Marrow Cells

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Long-term multilineage expression in peripheral blood from a Moloney murine leukemia virus vector after serial transplantation of transduced bone marrow cells

Blood ◽

10.1182/blood.v95.3.829.003k18_829_836 ◽

2000 ◽

Vol 95 (3) ◽

pp. 829-836 ◽

Cited By ~ 8

Author(s):

Timothy W. Austin ◽

Suzan Salimi ◽

Gabor Veres ◽

Franck Morel ◽

Heini Ilves ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Blood Cells ◽

Transgene Expression ◽

Bone Marrow Cells ◽

Leukemia Virus ◽

Moloney Murine Leukemia Virus ◽

Marrow Cells

Using a mouse bone marrow transplantation model, the authors evaluated a Moloney murine leukemia virus (MMLV)-based vector encoding 2 anti-human immunodeficiency virus genes for long-term expression in blood cells. The vector also encoded the human nerve growth factor receptor (NGFR) to serve as a cell-surface marker for in vivo tracking of transduced cells. NGFR+ cells were detected in blood leukocytes of all mice (n=16; range 16%-45%) 4 to 5 weeks after transplantation and were repeatedly detected in blood erythrocytes, platelets, monocytes, granulocytes, T cells, and B cells of all mice for up to 8 months. Transgene expression in individual mice was not blocked in the various cell lineages of the peripheral blood and spleen, in several stages of T-cell maturation in the thymus, or in the Lin−/loSca-1+ and c-kit+Sca-1+ subsets of bone marrow cells highly enriched for long-term multilineage-reconstituting activity. Serial transplantation of purified NGFR+c-kit+Sca-1+bone marrow cells resulted in the reconstitution of multilineage hematopoiesis by donor type NGFR+ cells in all engrafted mice. The authors concluded that MMLV-based vectors were capable of efficient and sustained transgene expression in multiple lineages of peripheral blood cells and hematopoietic organs and in hematopoietic stem cell (HSC) populations. Differentiation of engrafting HSC to peripheral blood cells is not necessarily associated with dramatic suppression of retroviral gene expression. In light of earlier studies showing that vector elements other than the long-terminal repeat enhancer, promoter, and primer binding site can have an impact on long-term transgene expression, these findings accentuate the importance of empirically testing retroviral vectors to determine lasting in vivo expression.

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Augmented Production of Interleukin-6 by Normal Human Osteoblasts in Response to CD34+ Hematopoietic Bone Marrow Cells In Vitro

Blood ◽

10.1182/blood.v89.4.1165 ◽

1997 ◽

Vol 89 (4) ◽

pp. 1165-1172 ◽

Cited By ~ 51

Author(s):

Russell S. Taichman ◽

Marcelle J. Reilly ◽

Rama S. Verma ◽

Stephen G. Emerson

Keyword(s):

Bone Marrow ◽

Interleukin 6 ◽

Transforming Growth Factor ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Cell Types ◽

Colony Stimulating Factor ◽

Cd34 Cells ◽

Stimulating Factor ◽

Marrow Cells

Abstract Based on anatomic and developmental findings characterizing hematopoietic cells in close approximation with endosteal cells, we have begun an analysis of osteoblast/hematopoietic cell interactions. We explore here the functional interdependence between these two cell types from the standpoint of de novo cytokine secretion. We determined that, over a 96-hour period, CD34+ bone marrow cells had no significant effect on osteoblast secretion of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, or transforming growth factor-β1 , but in some experiments minor increases in leukemia inhibitory factor levels were observed. However, when CD34+ bone marrow cells were cocultured in direct contact with osteoblasts, a 222% ± 55% (range, 153% to 288%) augmentation in interleukin-6 (IL-6) synthesis was observed. The accumulation of IL-6 protein was most rapid during the initial 24-hour period, accounting for nearly 55% of the total IL-6 produced by osteoblasts in the absence of blood cells and 77% of the total in the presence of the CD34+ cells. Cell-to-cell contact does not appear to be required for this activity, as determined by coculturing the two cell types separated by porous micromembranes. The identity of the soluble activity produced by the CD34+ cells remains unknown, but is not likely due to IL-1β or tumor necrosis factor-α, as determined with neutralizing antibodies. To our knowledge, these data represent the first demonstration that early hematopoietic cells induce the production of molecules required for the function of normal bone marrow microenvironments, in this case through the induction of hematopoietic cytokine (IL-6) secretion by osteoblasts.

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Host Conditioning With 5-Fluorouracil and kit-Ligand to Provide for Long-Term Bone Marrow Engraftment

Blood ◽

10.1182/blood.v89.7.2376 ◽

1997 ◽

Vol 89 (7) ◽

pp. 2376-2383 ◽

Cited By ~ 25

Author(s):

Ronald van Os ◽

Donald Dawes ◽

John M.K. Mislow ◽

Alice Witsell ◽

Peter M. Mauch

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Mixed Chimerism ◽

Kit Ligand ◽

Donor Bone Marrow ◽

Before And After ◽

Total Body ◽

Marrow Cells

Abstract Administration of kit-ligand (KL) before and after doses of 5-fluorouracil (5-FU) results in marrow failure in mice, presumably because of enhanced KL-induced cycling of stem cells, which makes them more susceptible to the effects of 5-FU. In attempt to capitalize on this effect on stem cells, we studied the ability of KL and 5-FU to allow stable donor engraftment of congenically marked marrow in a C57BL/6 (B6) mouse model. KL was administered subcutaneously at 50 μg/kg, 21 hours and 9 hours before and 3 hours after each of two doses of 5-FU (125 mg/kg) given 7 days apart to B6-recipients. Animals then received three injections of 107 congenic B6-Gpi-1a-donor bone marrow cells at 24, 48, and 72 hours after the second 5-FU dose. A separate group of animals received a single dose of either 1 × 107 or 3 × 107 donor marrow cells 24 hours after the last 5-FU dose. The level of engraftment was measured from Gpi-phenotyping at 1, 3, 6, and 8 months in red blood cells (RBCs) and at 8 months by phenotyping cells from the thymus, spleen, and marrow. Percent donor engraftment in RBCs appeared stable after 6 months. The percent donor engraftment in RBCs at 8 months was significantly higher in KL + 5-FU prepared recipients (33.0 ± 2.7), compared with 5-FU alone (18.5 ± 2.6, P < .0005), or saline controls (17.8 ± 1.7, P < .0001). In an additional experiment, granulocyte colony-stimulating factor (100 μg/dose) was added to a reduced dose of KL (12.5 μg/dose); engraftment was similar to KL alone. At 8 months after transplantation the levels of engraftment in other tissues such as bone marrow, spleen, and thymus correlated well with erythroid engraftment to suggest that multipotent long-term repopulating stem cells had engrafted in these animals. There are concerns for the toxicity of total body irradiation (TBI)- or busulfan-based regimens in young recipients of syngeneic or transduced autologous marrow who are transplanted for correction of genetic disease. In these recipients complete donor engraftment may not be needed. The results with KL and 5-FU are encouraging for the further refinement of non-TBI, nonbusulfan techniques to achieve stable mixed chimerism.

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In vitro proliferation of primitive hemopoietic stem cells supported by stromal cells: evidence for the presence of a mechanism(s) other than that involving c-kit receptor and its ligand.

Journal of Experimental Medicine ◽

10.1084/jem.176.2.351 ◽

1992 ◽

Vol 176 (2) ◽

pp. 351-361 ◽

Cited By ~ 44

Author(s):

H Kodama ◽

M Nose ◽

Y Yamaguchi ◽

J Tsunoda ◽

T Suda ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Hemopoietic Stem Cells ◽

In Vitro Proliferation ◽

Kit Receptor ◽

Colony Forming Units ◽

Marrow Cells

The preadipose cell line, PA6, can support long-term hemopoiesis. Frequency of the hemopoietic stem cells capable of sustaining hemopoiesis in cocultures of bone marrow cells and PA6 cells for 6 wk was 1/5.3 x 10(4) bone marrow cells. In the group of dishes into which bone marrow cells had been inoculated at 2.5 x 10(4) cells/dish, 3 of 19 dishes (16%) contained stem cells capable of reconstituting erythropoiesis of WBB6F1-W/Wv mice, indicating that PA6 cells can support the proliferation of primitive hemopoietic stem cells. When the cocultures were treated with an antagonistic anti-c-kit monoclonal antibody, ACK2, only a small number of day 12 spleen colony-forming units survived; and hemopoiesis was severely reduced. However, when the cocultures were continued with antibody-free medium, hemopoiesis dramatically recovered. To examine the proliferative properties of the ACK2-resistant stem cells, we developed a colony assay system by modifying our coculture system. Sequential observations of the development of individual colonies and their disappearance demonstrated that the stem cells having higher proliferative capacity preferentially survive the ACK2 treatment. Furthermore, cells of subclones of the PA6 clone that were incapable of supporting long-term hemopoiesis expressed mRNA for the c-kit ligand. These results suggest that a mechanism(s) other than that involving c-kit receptor and its ligand plays an important role in the survival and proliferation of primitive hemopoietic stem cells.

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