Peripheral Blood Lineages Research Articles

Lineage-specific Chimaerism Quantification after T-cell Depleted Peripheral Blood Stem Cell Transplantation

Patients that receive a T-cell depleted (TCD) hematopoietic stem cell transplantation (SCT) show higher risk of graft failure/rejection and of disease relapse than those that receive unmanipulated grafts. The purpose of the present investigation was to analyze the usefulness of chimaerism quantification in bone marrow (BM), peripheral blood (PB), and leukocyte lineages such as T lymphocytes (CD3+, both CD4+ and CD8+), B lymphocytes (CD19+) and myeloid cells (CD15+), for the early detection of graft failure/rejection episodes and disease relapse after TCD-PBSCT. Two of the ten (2/10) patients included in the study showed stable complete chimaerism (CC). The other 8/10 patients showed decreasing mixed chimaerism (MC) and 7 of them had either graft failure (n =1) /rejection (n =3) or disease relapse (n =3). In two patients relapsed from chronic myeloid leukemia, MC was observed in BM and PB, with higher percentages of autologous cells in BM, as well as in leukocyte lineages, with higher percentages of recipient cells in the myeloid lineage than in lymphocytes. Combined analysis of chimaerism and minimal residual disease allowed early diagnosis of relapse and successful rescue therapy with donor leukocyte infusions (DLI), before the onset of hematological relapse. Chimaerism analysis allowed early diagnosis of incipient graft rejection in 3 patients. These patients showed MC both in BM and PB, with greater percentages of recipient cells in PB. Analysis of leukocyte lineages showed higher percentages of autologous cells in T lymphocytes (mainly CD8+) than in B or myeloid cells. Two of these patients were successfully treated with DLI and recovered normal PB counts and BM cellularity, as well as CC. The graft versus recipient hemopoiesis effect harbored by the donor immunocompetent cells infused seems useful for the treatment of graft rejection, provided that an early diagnosis is made.

Muscle-derived hematopoietic stem cells are hematopoietic in origin.

It has recently been shown that mononuclear cells from murine skeletal muscle contain the potential to repopulate all major peripheral blood lineages in lethally irradiated mice, but the origin of this activity is unknown. We have fractionated muscle cells on the basis of hematopoietic markers to show that the active population exclusively expresses the hematopoietic stem cell antigens Sca-1 and CD45. Muscle cells obtained from 6- to 8-week-old C57BL/6-CD45.1 mice and enriched for cells expressing Sca-1 and CD45 were able to generate hematopoietic but not myogenic colonies in vitro and repopulated multiple hematopoietic lineages of lethally irradiated C57BL/6-CD45.2 mice. These data show that muscle-derived hematopoietic stem cells are likely derived from the hematopoietic system and are a result not of transdifferentiation of myogenic stem cells but instead of the presence of substantial numbers of hematopoietic stem cells in the muscle. Although CD45-negative cells were highly myogenic in vitro and in vivo, CD45-positive muscle-derived cells displayed only very limited myogenic activity and only in vivo.

Protection and in Vivo Selection of Hematopoietic Stem Cells Using Temozolomide, O6-Benzylguanine, and an Alkyltransferase-Expressing Retroviral Vector

Transfer of drug resistance genes to hematopoietic stem cells offers the potential to protect cancer patients from drug-induced myelosuppression and to increase the number of gene-modified cells by in vivo selection. In this study, a retroviral vector expressing both a P140K variant of human O6-methylguanine-DNA methyltransferase (MGMT) and an EGFP reporter gene was evaluated for stem cell protection in a murine transplant model. Mice transplanted with vector-transduced cells showed significant resistance to the myelosuppressive effects of temozolomide (TMZ), an orally administered DNA-methylating drug, and O6-benzylguanine (BG), a drug that depletes cells of wild-type MGMT activity. Following drug treatment, increases in EGFP+ peripheral blood cells were seen in all peripheral blood lineages, and secondary transplant experiments proved that selection had occurred at the stem cell level. In a second set of experiments in which transduced cells were diluted with unmarked cells, efficient stem cell selection was noted together with progressive marrow protection with repeated treatment courses. Altogether, these results show that P140K MGMT gene transfer can protect stem cells against the toxic effects of TMZ and BG and that this vector/drug system may be useful for clinical myeloprotection and for in vivo selection of transduced stem cells.

Use of the zebrafish (Danio rerio) to define hematopoiesis.

Hematopoiesis in the vertebrate is characterized by the induction of ventral mesoderm to form hematopoietic stem cells and the eventual differentiation of these progenitors to form the peripheral blood lineages. Several genes have been implicated in the differentiation and development of hematopoietic and vascular progenitor cells, yet our understanding of the discrete steps involved in the induction of these cells from the ventral mesoderm is still incomplete. One method of delineating these processes is based on the use of lower vertebrates. The zebrafish (Danio rerio) is an especially robust vertebrate system for both isolating and characterizing genes involved in these processes. Hematopoietic mutants have been generated with defects in many of the steps of both the primitive and definitive hematopoietic programs. Cloning of the genes that underlie these mutations should yield valuable details of hematopoiesis and may have therapeutic implications for bone marrow transplantation and stem cell gene therapy.

Use of the zebrafish (Danio rerio) to define hematopoiesis.

Hematopoiesis in the vertebrate is characterized by the induction of ventral mesoderm to form hematopoietic stem cells and the eventual differentiation of these progenitors to form the peripheral blood lineages. Several genes have been implicated in the differentiation and development of hematopoietic and vascular progenitor cells, yet our understanding of the discrete steps involved in the induction of these cells from the ventral mesoderm is still incomplete. One method of delineating these processes is based on the use of lower vertebrates. The zebrafish (Danio rerio) is an especially robust vertebrate system for both isolating and characterizing genes involved in these processes. Hematopoietic mutants have been generated with defects in many of the steps of both the primitive and definitive hematopoietic programs. Cloning of the genes that underlie these mutations should yield valuable details of hematopoiesis and may have therapeutic implications for bone marrow transplantation and stem cell gene therapy.

Molecular insights into early hematopoiesis.

During embryogenesis, a pool of pluripotential, self-renewing hematopoietic stem cells is specified from undifferentiated ventral mesoderm. These cells proliferate and differentiate to yield all the peripheral blood lineages. Although the mechanisms involved in the commitment and differentiation of multipotential progenitors have been well documented, little focus has been directed to the molecular events underlying the formation of hematopoietic stem cells. In this review we discuss the recent advances made in defining a bone morphogenetic protein-responsive signal cascade that regulates the earliest phase of blood formation during embryogenesis.

AM218, a new polyanionic polysaccharide, induces radioprotection in mice when administered shortly before irradiation

We have shown that the polyanionic polysaccharide AM218 improves the survival rate of the potentially lethally irradiated mice. This radioprotective effect was highly dependent on the administration schedule, the most efficient protocol being that in which the drug was given shortly before irradiation. The haematopoietic implications in the pharmacological action of AM218 were confirmed by the improved recovery in the three peripheral blood lineages observed in the AM218-treated mice. However, because of a marked increase observed in the number of white blood cells during the period of highest mortality of the control irradiated mice, effects on the neutrophil lineage may account for the effects mediated by AM218 in the irradiated mice. Both in vitro and in vivo treatment with AM218 before irradiation improved the survival rate of CFU-GM progenitors, while no effects were observed on the CFU-S pool. This led us to postulate that the improved survival rate of the committed progenitors, at least the granulocyte-macrophage progenitors, accounts for the radioprotective capacity of AM218.

Differential Expression of Receptors for Interleukin-3 on Subsets of CD34-Expressing Hematopoietic Cells of Rhesus Monkeys

The target cell specificity of interleukin-3 (IL-3) was examined by flow cytometric analysis of IL-3 receptor (IL-3R) expression on rhesus monkey bone marrow (BM) cells using biotinylated IL-3. Only 2% to 5% of unfractionated cells stained specifically with the biotinylated IL-3 and most of these cells were present within the CD34+ subset. IL-3Rs were detected on small CD34dull/RhLA-DRbright/CD10+/CD27+/CD2-/++ +CD20- cells, which probably represent B-cell precursors. IL-3R+ CD34- BM cells, which were detected at low frequencies, consisted of small CD20dull/surface-IgM+/RhLA-DR+ cells. These cells represented immature B lymphocytes, whereas CD20bright mature B cells were IL-3R-. The highest IL-3R levels were detected on CD34dull/RhLA-DRbright blast-like cells. These cells differentiated into monocytes, neutrophils, and basophils after IL-3 and/or granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation in vitro. The CD34bright/IL-3R- subset contained all clonogenic erythroid and myeloid progenitors (burst-forming unit-erythroid and colony-forming unit-culture), whereas CD34bright/IL-3Rdull cells differentiated into monocytes, neutrophils, and erythroid cells after shorter culture periods. This finding showed that IL-3R expression increases during monocyte and granulocyte differentiation. Results of three-color experiments indicated that IL-3Rs are expressed on CD34bright/RhLA-DRbright cells as well as on CD34bright/RhLA-DRdull cells, with the latter population expression approximately twofold to threefold lower IL-3R levels. A large fraction (> 30%) of single-cell/well-sorted CD34bright/RhLA-DRdull cells formed multilineage colonies after 2 to 4 weeks of stimulation with IL-3, GM-CSF, Kit ligand, and IL-6. Individual colonies contained cells that still expressed CD34 as well as differentiated monocytes, granulocytes, and erythroid cells. These results confirmed that the CD34bright/RhLA-DRdull subset was enriched for immature, multipotent progenitor cells, whereas the CD34bright/RhLA-DRbright population mainly contained lineage-committed precursors. The results are consistent with the concept that IL-3Rs are induced at very early stages of hematopoiesis, as identified by high expression of CD34 and low expression of RhLA-DR. IL-3R expression continues to be low during differentiation into lineage-committed progenitors; gradually increases on differentiating progenitor cells for B cells, granulocytes, monocytes, and, possibly also, erythrocytes; but finally declines to undetectable levels during terminal differentiation into mature cells of all lineages in peripheral blood, with the exception of basophils.(ABSTRACT TRUNCATED AT 400 WORDS)

Varying populations of CD59-negative, partly positive, and normally positive blood cells in different cell lineages in peripheral blood of paroxysmal nocturnal hemoglobinuria patients.

CD59-antigen expression on the surface membranes of erythrocytes, granulocytes, monocytes, lymphocytes, and platelets was determined by flow cytometry in 34 healthy controls and 17 patients with paroxysmal nocturnal hemoglobinuria (PNH). In all PNH patients, CD59-negative erythrocytes accounted for > 10% of the total erythrocyte population. Two erythrocyte populations (CD59-negative and normally positive or CD59-negative and partly positive), three populations (CD59-negative, partly positive, and normally positive), and one population (CD59-negative) were demonstrated in ten, six, and one patients, respectively. However, CD59-negative granulocytes did not account for > 10% of the total granulocytes in two patients, and one of them had only a CD59 normally positive granulocyte population. A particular granulocyte population extended over both CD59-negative and partly positive areas was shown in two patients. Two populations (CD59-negative and normally positive) and one population (CD59-negative) were demonstrated in monocytes and lymphocytes. CD59-negative lymphocytes accounted for > 50% of the total lymphocytes in only two patients. Three patients had a CD59 normally positive lymphocyte population. Percentages of CD59-positive platelet population in normal controls were widely various. Therefore, it was usually difficult to discriminate between PNH-affected and normal platelets. Thus, the flow cytometric profiles of CD59-antigen expression varied not only between PNH patients but between cell lineages. The present results and our prior study indicate that CD59 flow cytometry using erythrocytes and granulocytes is most suitable for diagnosing PNH.

ES cells have only a limited lymphopoietic potential after adoptive transfer into mouse recipients

While hematopoietic stem cells from adult and fetal stages of murine development are capable of long term reconstitution of all mature blood lineages in vivo, embryonic hematopoietic stem cell repopulation in vivo has proved difficult. It is thought that there are many fewer hematopoietic stem cells in the embryo than in the fetal/adult stages of mouse development and that these cells possess a different developmental potential. One source of such cells are embryonic stem (ES) cells which can differentiate into most mature blood lineages in vitro. We have therefore used transplantation of differentiated ES cells to assess the hematopoietic potential of embryonic hematopoietic cells in vivo. We demonstrate here that precursors obtained from in vitro cultures of normal ES cells can contribute only to restricted and limited hematopoiesis in a mouse without leading to tumour formation. Repopulation occurs for greater than 6.5 months at levels ranging from 0.1% to 6% in B and T cell lineages in peripheral blood. In contrast to in vitro colony data demonstrating the myeloid lineage developmental potential of ES cells, no donor-derived myeloid repopulation was observed in CFU-S assays and no macrophage and mast cells were found in long term repopulated recipients. Thus, the hematopoietic potential of ES cells in vivo is limited to low levels of repopulation and is restricted to the lymphoid lineage.

Basic Fibroblast Growth Factor Expression in Human Bone Marrow and Peripheral Blood Cells

We have shown previously that basic fibroblast growth factor (bFGF) is a mitogen for human bone marrow (BM) stromal cells and that bFGF stimulates myelopoiesis in primary BM cultures. In this article, we demonstrate the presence of bFGF in two cell lineages in human BM and peripheral blood as well as the deposition of bFGF into the extracellular matrix of BM stromal cell cultures. In immunofluorescence experiments on BM and peripheral blood smears, megakaryocytes and platelets stained strongly for bFGF, whereas weaker staining was observed in immature and mature cells of the granulocyte series. The presence of bFGF in platelets was confirmed by enzyme-linked immunosorbent assay as well as by immunoprecipitation followed by immunoblotting. bFGF was synthesized by BM stromal cell cultures and was found either cell associated or localized in the nucleus and the nucleoli, and its location was dependent on the fixation procedure used. Addition of exogenous bFGF to stromal cells showed the presence of extracellular binding molecules for this cytokine. bFGF could be released from these sites by soluble heparin or phosphatidylinositol-specific phospholipase C. This study supports the role of bFGF as a stromal cell mitogen and stimulator of myelopoiesis. The data indicate that the stromal cells produce bFGF and that their extracellular matrix can serve as a reservoir for this growth factor. In addition, the results suggest a possible involvement of bFGF in platelet function as well as in megakaryocytopoiesis.

Basic fibroblast growth factor expression in human bone marrow and peripheral blood cells

We have shown previously that basic fibroblast growth factor (bFGF) is a mitogen for human bone marrow (BM) stromal cells and that bFGF stimulates myelopoiesis in primary BM cultures. In this article, we demonstrate the presence of bFGF in two cell lineages in human BM and peripheral blood as well as the deposition of bFGF into the extracellular matrix of BM stromal cell cultures. In immunofluorescence experiments on BM and peripheral blood smears, megakaryocytes and platelets stained strongly for bFGF, whereas weaker staining was observed in immature and mature cells of the granulocyte series. The presence of bFGF in platelets was confirmed by enzyme-linked immunosorbent assay as well as by immunoprecipitation followed by immunoblotting. bFGF was synthesized by BM stromal cell cultures and was found either cell associated or localized in the nucleus and the nucleoli, and its location was dependent on the fixation procedure used. Addition of exogenous bFGF to stromal cells showed the presence of extracellular binding molecules for this cytokine. bFGF could be released from these sites by soluble heparin or phosphatidylinositol- specific phospholipase C. This study supports the role of bFGF as a stromal cell mitogen and stimulator of myelopoiesis. The data indicate that the stromal cells produce bFGF and that their extracellular matrix can serve as a reservoir for this growth factor. In addition, the results suggest a possible involvement of bFGF in platelet function as well as in megakaryocytopoiesis.

Clonal and systemic analysis of long-term hematopoiesis in the mouse.

We have analyzed the temporal in vivo fate of 142 individual stem cell clones in 63 reconstituted mice. Long-term sequential analyses of the four major peripheral blood lineages, obtained from animals engrafted with genetically marked stem cells, indicate that developmental behavior is primarily a function of time. As such, the first 4-6 months post-engraftment is characterized by frequent fluctuations in stem cell proliferation and differentiation behavior. Gradually, a stable hematopoietic system emerges, dominated by a small number of totipotent clones. We demonstrate that single stem cell clones are sufficient to maintain hematopoiesis over the lifetime of an animal and suggest that mono- or oligoclonality may be a hallmark of long-term reconstituted systems. A model is proposed, wherein lineage-restricted differentiation and dramatic clonal flux are consequences of mechanisms acting on an expanding pool of totipotent cells and are not indicative of intrinsically distinct stem cell classes.