Hematopoietic Progenitor Colony Research Articles

Effects of plastic-adherent dermal multipotent cells on peripheral blood leukocytes and CFU-GM in rats

Multipotent cells were isolated from the dermis of newborn rats by their adherence to plastic culture dishes. Morphological observations showed that most of these elements were fibroblast-like cells described as cultured dermal multipotent cells (DMCs). These cells express CD90, CD44, CD59, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). They display the capacity to differentiate into cells with the phenotypic properties of osteocytes, adipocytes, chondrocytes, or neurons depending upon the specific inducing media. The confluent dermal multipotent cell layer supported and promoted the in vitro growth of colony-forming unit of granulocyte/macrophage progenitors (CFU-GM) as well as hematopoietic progenitor colonies. Transplantation of dermal multipotent cells into sublethally irradiated rats led to a significant increase of peripheral blood white cells nucleated cells, and CFU-GM colonies in the bone marrow. Fluorescence in situ hybridization analysis using a Y-chromosome–specific probe showed that dermal multipotent cells could engraft into bone marrow. However, they failed to differentiate into CD45-positive hematopoietic cells and to repopulate the hematopoietic system of lethally irradiated rats. These results suggest that plastic-adherent DMCs may at least represent an alternative source of mesenchymal stem cells to restore the marrow microenvironment and promote the survival, engraftment, and proliferation of hematopoietic cells.

In vitro inhibition of murine hematopoietic progenitors and stromal cells by vinorelbine.

Hematopoietic progenitor colony assays were used to establish the effects of the vinca alkaloid vinorelbine (VRB) on murine bone marrow. The in vitro growth of colony-forming units-granulocyte/macrophage (CFU-GM), burst forming units-erythroid (BFU-E) and colony-forming units-mix (CFU-mix) was dose-dependently inhibited by VRB. The highest dose assayed (0.02 microg/ml) suppressed all of the different progenitor cells by 100%. A comparison of the dose-response curves showed that CFU-GM, BFU-E, and CFU-mix exhibited similar-patterns of sensitivity to the cytotoxic action of VRB. Long-term bone marrow cultures have provided a valuable in vitro model for studying the role of the microenvironment of bone marrow. Cellularity of stromal layers was reduced with increasing doses of VRB. The appearance of these layers was altered minimally with the lowest dose used; a gradual loss of cellularity was seen in cultures exposed to 0.05 and 0.075 microg/ml; and a marked loss at the dose of 0.1 microg/ml. Our results show that VRB has an important effect on hematopoietic progenitors at the highest dose tested, while the stromal cells were not affected at a similar dose (0.025 microg/ml), suggesting that the stroma is more resistant to this drug.

Cloning, biological characterization and high-level expression of rat interleukin-3 using recombinant adenovirus: description of a new splicing variant.

In the present study, we describe the cloning and sequence analysis of rat IL-3. Two different mRNA isoforms were isolated after transfection of COS cells with the cytokine genomic sequences. One of the isoforms has been predicted before by Cohen et al. (1986), and the other one is identical except that it encodes a protein with an insertion of three amino acids at position 56. As names for the two isoforms, we propose IL-3alpha for the predicted and IL-3beta for the novel molecule. IL-3beta mRNA was detected as the predominant isoform in rat lymphocytes in vivo. High levels of the cytokine were obtained after infection of human cells (A549) with a recombinant adenovirus harboring rIL-3beta cDNA (IG.Ad.CMV.IL-3beta). The biological properties of the IL-3beta protein were tested in a FDC-P1 proliferation assay and in a hematopoietic progenitor colony forming assay. To assess in-vivo bioactivity, lysed 293 cells containing IG.Ad.CMV.rIL-3beta virus were injected subcutaneously into F344 rats. Stimulation of hematopoiesis and leucocytosis were observed during the treatment. After subcutaneous injections of the lysed adeno-producer cells in mice, the only effect observed was a cellular infiltration at the site of injection, confirming the poor cross-reactivity between the two species. The biological properties in vitro and in vivo demonstrate that the cDNA sequences of IL-3beta presented here encode active rat IL-3 protein.

Assessment of mitomycin C sensitivity in Fanconi anemia complementation group C gene (Fac) knock-out mouse cells.

Fanconi anemia (FA) is a genetic disorder defined by cellular hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). MMC causes increased FA cell death, chromosome breakage, and accumulation in the G2 phase of the cell cycle. Recently, Fanconi anemia complementation group C (fac) gene knock-out mice have been developed, and SV40-transformed fibroblasts were established from fac homozygous knock-out (-/-), heterozygous (+/-), and wild-type mice (+/+). MMC sensitivity of these cell lines was assessed by three methods: colony-formation assay in the presence of MMC, chromosome breakage, and cell cycle analysis to detect G2 phase arrest. The fac knock-out fibroblasts (-/-) showed a significantly higher sensitivity to MMC than did fibroblasts from wild-type (+/+) or heterozygous (+/-) mice (three experiments). In addition, we analyzed hematopoietic progenitor colony assays of bone marrow cells from fac knock-out (-/-) and heterozygous (+/-) mice. CFU-E, BFU-E, and CFU-GM colony formation from fac nullizygous mouse progenitors was markedly diminished by MMC when compared to growth of progenitors from heterozygous mice. These results show that fac knock-out mouse cells mimic the behavior of human FA-C patient cells in terms of MMC hypersensitivity. The fac knock-out mouse may be used to model some aspects of human FA and should be useful for understanding the function of the FAC protein.

A Patient With Paroxysmal Nocturnal Hemoglobinuria Bearing Four Independent PIG-A Mutant Clones

AbstractParoxysmal nocturnal hemoglobinuria (PNH) is characterized by clonal blood cells that are deficient in the surface expression of glycosylphosphatidylinositol-anchored proteins due to somatic mutation in the X-linked gene PIG-A. In some patients, more than one abnormal clone may be present. Analysis of bulk DNA/RNA from granulocytes has been useful in identifying the predominant PIG-A mutation in each patient. However, it is often not useful in determining the presence of minor clones. Many patients have cells with partial deficiency. Here, we analyzed the PIG-A gene in two B-cell lines bearing complete or partial deficiencies, cells of hematopoietic progenitor colonies and peripheral blood granulocytes from the same patient. We found that two B-cell lines had different mutations, the granulocytes contained at least two mutants, and the hematopoietic progenitors contained four mutants. Three of the four were shared by B cells and/or granulocytes whereas the other one was found only in the hematopoietic progenitors. The partial deficiency was caused by a point mutation near an alternative splice site within exon 2 that resulted in partial decreases of activity and quantity of the full-length transcript. These results further show the oligoclonal nature of PNH and differences in extent of expansion among mutant clones.

In vitro potency of inhibition by antiviral drugs of hematopoietic progenitor colony formation correlates with exposure at hemotoxic levels in HIV positive humans

In vitro potency of inhibition by antiviral drugs of hematopoietic progenitor colony formation correlates with exposure at hemotoxic levels in HIV positive humans

Preclinical biology of interleukin 11: a multifunctional hematopoietic cytokine with potent thrombopoietic activity.

Interleukin 11 (IL-11) is a multifunctional hematopoietic cytokine which was originally identified as a factor produced by an IL-1-stimulated primate stromal cell line. The in vitro biological activities of recombinant human (rHu)IL-11 result predominantly from synergistic interactions with other growth factors. In combination with other cytokines, rHuIL-11 has been shown to support the formation of primitive hematopoietic and lymphohematopoietic progenitor colonies from bone marrow, to promote erythroid burst formation and to stimulate both early and late stages of megakaryocyte proliferation and differentiation. rHuIL-11 is biologically active in mice, rats, dogs and primates when administered as a single agent in vivo. The predominant effect of rHuIL-11 in naive mice was on cells of the megakaryocytic lineage, increasing the number of bone marrow megakaryocyte progenitors, stimulating megakaryocyte endoreplication and increasing peripheral platelet counts in a dose-dependent fashion. Similar megakaryocytic stimulatory activity was seen in nonhuman primates treated with rHuIL-11 where platelet counts were increased by as much as 300%. In several models of severe myelosuppression induced by chemotherapy and/or irradiation and in bone marrow transplant models, there were multilineage hematopoietic stimulation following rHuIL-11 treatment. In these models, accelerated recovery of platelets was a consistent observation, while some models show enhanced neutrophil and red blood cell recovery as well. These results from preclinical studies confirm the broad spectrum of biological activities exhibited by rHuIL-11 in vitro, and suggest that this cytokine may be an effective agent in the treatment of myelosuppression and thrombocytopenia associated with cancer chemotherapy and bone marrow transplantation.

Lymphokines: Enhancement by Granulocyte-Macrophage and Granulocyte Colony-Stimulating Factors of Neonatal Myeloid Kinetics and Functional Activation of Polymorphonuclear Leukocytes

Colony-stimulating factors, such as the granulocyte-macrophage and the granulocyte colony-stimulating factors (GM-CSF and G-CSF), are glycoproteins with biologic specificity defined by their ability to support proliferation and differentiation of hematopoietic cells of various lineages. Their physiologic activities include stimulation and proliferation of early stem cell precursors and functional activation of mature peripheral effector cells. Recently produced recombinant human (rh) GM-CSF and G-CSF have been demonstrated to regulate hematopoietic neutrophil progenitor colony growth; to stimulate the release of bone marrow neutrophil storage pools; and to prime mature effector functions, including chemotaxis, oxidative metabolism, phagocytosis, C3bi receptor expression, and antibody-dependent cytotoxicity in adults. We examined the effects of rh-GM-CSF on priming superoxide release and chemotaxis of neonatal (cord) polymorphonuclear leukocytes (PMNs) and of rh-G-CSF and rh-GM-CSF on bone marrow neutrophil egress in the neonatal rat. A time-response evaluation of the effect of rh-GM-CSF revealed enhanced release of superoxide by PMNs. PMN chemotaxis also was enhanced by rh-GM-CSF, with a maximal response occurring earlier than enhanced superoxide release. Intraperitoneal administration of rh-G-CSF or rh-GM-CSF to 1-day-old rats resulted in significant increases in white blood cell counts and significant early neutrophilia. Bone marrow examination revealed that the neutrophilia was secondary to egress and mild depletion of the neutrophil storage pool but that the neutrophil storage pool later returned to normal. These preliminary studies suggest that rh-GM-CSF and rh-G-CSF prime neonatal effector function and induces significant PMN egress and neutrophilia.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of hematopoietic progenitor colony growth by a monoclonal antibody against the transferrin receptor: Comparison of unconjugated antibody with an immunotoxin containing recombinant ricin a chain

We studied an immunotoxin consisting of recombinant ricin A chain (rRA) conjugated to 454A12 MoAb, a monoclonal antibody which recognizes an epitope on the human transferrin receptor, and compared the ability of 454A12 MoAb-rRA immunotoxin to inhibit the growth of erythroid burst-forming units (BFU-e) and myeloid colony-forming units (CFU-c) with unconjugated 454A12 MoAb. A significant reduction in BFU-e colony growth was observed at 0.001 microgram/ml of 454A12 MoAb-rRA versus 0.1 microgram/ml of unconjugated 454A12 MoAb (p = 0.005). Comparison of the effects of 454A12 MoAb-rRA and 454A12 MoAb on myeloid colony development gave markedly different results. Unconjugated antibody had no effect on CFU-c colony growth; in contrast, 0.01 microgram/ml of 454A12 MoAb-rRA reduced the number of colonies from 139 per 1 X 10(5) to 75 per 1 X 10(5) cells plated (p = 0.0005). No myeloid progenitor colonies developed at 0.1 microgram/ml of immunotoxin. These observations suggest that 454A12 MoAb-rRA inhibits growth by a potent, ricin A chain-mediated toxic effect on any proliferating cells expressing transferrin receptors, whereas the 454A12 MoAb exerts a selective inhibitory effect primarily on erythroid progenitors by perturbing the transferrin cycle. While growth factor receptors expressed on hematopoietic cells represent promising targets for immunotoxin therapy, our data indicate that an immunotoxin could inhibit cellular proliferation by a different mechanism than the corresponding unconjugated MoAb. Depending on the antibody used, these differences may be important in trials using immunotoxins for in vivo treatment or in vitro purging of malignant hematopoietic cells.

Circulating Mononuclear Cells from Pure Red Cell Aplasia of Chronic Lymphocytic Leukemia Suppress in vitro Erythropoiesis

In vitro studies were performed in a patient with B-cell chronic lymphocytic leukemia who developed pure red cell aplasia (CLL-PRCA). The patient's irradiated circulating mononuclear blood cells and supernatant markedly inhibited normal marrow erythroid (but not granulocyte-monocyte) progenitor colony proliferation. In contrast, irradiated peripheral blood mononuclear cells and supernatant obtained from a B-CLL patient (Rai stage III) and from a hematologically normal donor, did not affect hematopoietic progenitor colony growth. These findings suggest that the anemia of CLL-PRCA evolves different mechanisms of those causing anemia in CLL, and is mediated through cellular and secretory mechanisms.

Differentiation in vitro of T3+ large granular lymphocytes with characteristic cytotoxic activity from an isolated hematopoietic progenitor colony.

Blast colonies were developed by rIL-3 from the spleen cells of mice pretreated with 5-fluorouracil (5-FU) in the methylcellulose cultures. When such IL-3-induced blast colonies were individually lifted up and recultured in the presence of rIL-3 and recombinant erythropoietin (rEpo), a variety of hematopoietic colonies developed from every single colony, including neutrophils, macrophages, eosinophils, megakaryocytes, mast cells, and erythroblasts. The results indicated that IL-3-induced blast colonies consisted of multipotential hematopoietic progenitor cells. By culturing individual IL-3-induced blast colonies in the presence of rIL-2 and irradiated peritoneal macrophages, on the other hand, the proliferation of homogeneous lymphoid cells was observed in 5 of 24 wells, each of which received a single blast colony. Morphologically, they were typical large granular lymphocytes (LGL), and thus it was indicated that LGL could be differentiated directly from the hematopoietic progenitor cells utterly in vitro by rIL-2 and accessory macrophages. From one of these culture wells, a continuous LGL line, IL3B1, was successfully obtained. The proliferation of IL3B1 was dependent on IL-2 in the presence of accessory macrophages, but they no longer responded to IL-3, nor to another T cell growth factor, IL-4. Flow cytometric analysis indicated that the phenotype of IL3B1 was Thy-1+,T3+,L3T4-,Lyt-2-,T200+ Asialo GM1+, whereas that of original IL-3-induced blast cells was Thy-1+,T3-,L3T4-,Lyt-2-,B220-. The results suggested that the expression of T3 molecules was induced in the process of LGL differentiation from the hematopoietic progenitor cells in vitro. Conforming to this, it was revealed that both gamma and beta chain genes of the TCR were rearranged in IL3B1. Northern blot analysis indicated that IL3B1 had abundant mRNA for gamma chain, while mRNA for beta chain was rather faint. Functionally, IL3B1 exhibited typical NK-patterned cytotoxic activity against a panel of tumor cell targets. In addition, they showed significant cytotoxic activity against normal bone marrow cells, as well as various factor-dependent myelogenous progenitor cell lines, all of which were resistant to endogenous NK activity of the fresh spleen cells. These results indicated that at least a set of T3+ LGL with rearranged TCR genes could be directly differentiated from isolated hematopoietic progenitor cells in vitro. Results also suggested that such a prethymically differentiated subset of T-lineage lymphocytes, namely T3+ double-negative LGL, had particular cytotoxic activity in addition to conventional NK activity, which might well contribute to feedback regulation of hematopoiesis.

GENETIC MANIPULATION OF HUMAN HEMATOPOIETIC CELLS WITH RETROVIRUS VECTORS

We transferred new genetic information into human cell lines and hematopoietic stem cells using retrovirus vectors. These vectors, containing the dominant selectable gene (neo) for resistance to the antibiotic G418, were introduced into amphotropic helper viruses to achieve a high frequency transduction into human cells. Human cell lines or freshly obtained human marrow cells were co-cultivated with irradiated vector producing cells, and cultured in clonogenic assays with and without G418. Survival curves of non-transfected cells with increasing concentrations of G418 resulted in a dose-dependent inhibition of colony growth with a final dose of 500 to 800 μg/ml. Transfected promyelocytic leukemia (HL-60), erythroleukemia (OCI-M2) and different lymphoid cell lines with N2 and SV(X) vectors, revealed G418 resistant colonies in frequencies ranging from 3.2 to 44%. Similar frequencies were found with human marrow cells. Transfection with either N2 or NEOμ vectors produced G418 resistant hematopoietic progenitor colonies of the erythroid (BFU-E) granulocyte-macrophage (CFU-GM), and mixed (GFU-GEMM) lineages in frequencies ranging from 8 to 40% after 14 days in culture. We conclude that selected retrovirus vectors can be successfully used to insert new genetic information into pluripotent and committed human hematopoietic progenitor cells.

In vitro and in vivo effects of deferoxamine in neonatal acute leukemia

A six week old infant with acute leukemia failed to attain remission with chemotherapy. Because we previously demonstrated that the iron chelator deferoxamine (DFO) has antiproliferative properties and modulatory effects on cell differentiation, a protocol was designed for in vitro study and for clinical use in the patient. At diagnosis, blast cells were morphologically undifferentiated, had nondiagnostic cytochemistry, showed an abnormal karyotype (t[4;11]), expressed markers of B cell lineage, and demonstrated C mu gene rearrangement. Tissue culture of marrow or blood cells yielded colonies of leukemic blasts. Increasing concentrations of DFO produced a dose-dependent suppression of patient's blast colony growth in vitro, and blasts within colonies showed a marked change in surface antigen expression from lymphoid to myelomonocytic markers, became monocytic in appearance, and developed intense staining for nonspecific esterase. When DFO was given intravenously to the patient as a single agent for 48 hours, blasts no longer expressed lymphoid antigens and became strongly positive for myelomonocytic markers, identical to the in vitro findings. Intravenous DFO halted rising peripheral blood blast cell numbers and allowed a several-fold increase in normal hematopoietic progenitor colony growth. When combined with low-dose cytosine arabinoside in the treatment protocol, DFO caused striking leukemic cytoreduction. Our findings indicate that DFO has antileukemic properties by virtue of its effects on proliferation and differentiation, and they prompt further experimental and clinical studies with this agent.

In Vitro and In Vivo Effects of Deferoxamine in Neonatal Acute Leukemia

A six week old infant with acute leukemia failed to attain remission with chemotherapy. Because we previously demonstrated that the iron chelator deferoxamine (DFO) has antiproliferative properties and modulatory effects on cell differentiation, a protocol was designed for in vitro study and for clinical use in the patient. At diagnosis, blast cells were morphologically undifferentiated, had nondiagnostic cytochemistry, showed an abnormal karyotype (t[4;11]), expressed markers of B cell lineage, and demonstrated C mu gene rearrangement. Tissue culture of marrow or blood cells yielded colonies of leukemic blasts. Increasing concentrations of DFO produced a dose-dependent suppression of patient's blast colony growth in vitro, and blasts within colonies showed a marked change in surface antigen expression from lymphoid to myelomonocytic markers, became monocytic in appearance, and developed intense staining for nonspecific esterase. When DFO was given intravenously to the patient as a single agent for 48 hours, blasts no longer expressed lymphoid antigens and became strongly positive for myelomonocytic markers, identical to the in vitro findings. Intravenous DFO halted rising peripheral blood blast cell numbers and allowed a several-fold increase in normal hematopoietic progenitor colony growth. When combined with low-dose cytosine arabinoside in the treatment protocol, DFO caused striking leukemic cytoreduction. Our findings indicate that DFO has antileukemic properties by virtue of its effects on proliferation and differentiation, and they prompt further experimental and clinical studies with this agent.