CD45 isoform expression on human haemopoietic cells at different stages of development.

Alternate splicing and glycosylation produce multiple CD45 isoforms which are selectively expressed on the surface of cells of the haemopoietic system. The expression of CD45RA, CD45RB and CD45RO on CD34+ and CD34- haemopoietic cells from umbilical cord blood, bone marrow and fetal liver were studied by flow cytometry. CD34+ subpopulations defined by CD45 isoform expression were sorted from bone marrow and tested in long-term culture assays.

Transforming growth factor beta 1 is an inducer of erythroid differentiation.

Normal human bone marrow cells, highly enriched for burst-forming units-erythroid (BFU-E), were cultured in serum-free medium, in the presence and absence of various factors, to investigate the mechanisms involved in regulating erythroid differentiation. In cultures containing interleukin 3 (IL-3), Steel factor (SF), and erythropoietin (Ep), benzidine-positive erythroblasts first became detectable on day 6. Their numbers then rapidly increased until, by day 16, > 99% of the cells, which were 20,000-fold amplified over input numbers, were benzidine-positive.

Autografting with cultured marrow in chronic myeloid leukemia: results of a pilot study.

Incubation of chronic myeloid leukemia (CML) marrow for 10 days in vitro causes a marked and selective loss of very primitive Philadelphia chromosome (Ph)+ as compared with Ph- progenitors. We have autografted 22 patients with CML (16 in first chronic phase [group 1] and 6 with more advanced disease [group 2]) with marrow treated in this way to facilitate restoration of Ph- hematopoiesis after intensive therapy. Hematologic recovery to greater than 0.

Expression of interleukin-1 beta gene in candidate human hematopoietic stem cells.

Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect interleukin-1 beta (IL-1 beta) mRNA in candidate human hematopoietic stem cells. The cells, obtained from adult bone marrow (BM) or umbilical cord blood, had a CD34+ CD45RAlo CD71lo phenotype and were further fractionated into CD38+ and CD38- or Thy-1+ and Thy-1- subpopulations. The purity of these fractions was always more than 99%.

Kinetics of committed and primitive blood progenitor mobilization after chemotherapy and growth factor treatment and their use in autotransplants.

Peripheral blood cells (PBCs) collected by leukapheresis after progenitor mobilization with chemotherapy and growth factors have been used successfully to replace marrow autografts in protocols requiring stem-cell support. Moreover, such transplants are often associated with more rapid recovery of blood cell counts than is routinely achieved with bone marrow. While conditions that mobilize colony-forming cells (CFCs) into the circulation are becoming increasingly well characterized, little information is available as to how these or other mobilizing treatments may influence the release of more primitive cells into the peripheral blood.

Thy-1 expression is linked to functional properties of primitive hematopoietic progenitor cells from human umbilical cord blood.

We have previously shown that the most primitive human hematopoietic cells are included within a cell subpopulation expressing high levels of CD34 and low or undetectable levels of CD45RA and CD71. In this study, cord blood cells with this phenotype were sorted and further separated based on their expression on the Thy-1 antigen. The proliferation and differentiation of the purified cell fractions in response to a mixture of hematopoietic cytokines was analyzed in serum- and stroma-free liquid cultures.

Cytokines acting early in human haematopoiesis.

In long-term cultures (LTC) of human haematopoietic cells, primitive progenitors termed LTC-initiating cells can be maintained for several months and will differentiate to produce clonogenic cells and mature granulocytes and macrophages when provided with a supportive feeder layer of adherent mesenchymal cells. Primitive haematopoietic cells become associated with this feeder layer and their proliferative status and differentiation are regulated by their interaction with these feeder cells and the growth factors they produce. Both positive and negative regulators are generated in LTC and the balance between these diverse factors is readily manipulated by both direct and indirect mechanisms which appear to operate in a localized fashion.

Age-related decline in proliferative potential of purified stem cell candidates.

Recent studies in our laboratory have shown striking differences in the functional properties of candidate hematopoietic stem cells purified from fetal, neonatal, and adult human tissues. These differences include the ability to produce CD34+ cells, the turnover rate, and the fraction of cells that respond to a mixture of cytokines. All these parameters decrease with the age of the cell donor, and some of these observations are summarized here.

Selective separation of cells using magnetic colloids.

We have developed a reliable, purely immunological means of quantitatively labeling cells with magnetic colloidal dextran iron. Labeled cells can be efficiently separated with a high gradient magnetic filter inside a magnetic field. Separation conditions and filter design can be changed to accommodate large and small scale positive or negative selection.

Amplification of Sca-1+ Lin- WGA+ cells in serum-free cultures containing steel factor, interleukin-6, and erythropoietin with maintenance of cells with long-term in vivo reconstituting potential.

Normal murine bone marrow (BM) cells were sorted on the basis of low forward and orthogonal light scatter properties, Sca-1 expression (Sca-1+), lack of staining with a cocktail of mature hematopoietic lineage markers (Lin-), and binding of wheat germ agglutinin (WGA+). This approach allowed the reproducible isolation of a very small subpopulation (0.037% +/- 0.

Lineage commitment in human hemopoiesis involves asymmetric cell division of multipotent progenitors and does not appear to be influenced by cytokines.

Different models have been proposed to explain lineage commitment in hemopoiesis. Some suggest that lineage commitment occurs in a stochastic manner without the direct influence of extracellular factors; others postulate that cytokines determine whether multipotent cells will become erythroid or granulocyte/macrophage progenitors. In the present study, the patterns of proliferation and differentiation of individually sorted human cord blood-derived primitive hemopoietic cells (highly enriched for multipotent progenitors) were analyzed in a serum-free culture system supplemented with different cytokine combinations.

Characterization of functionally distinct subpopulations of CD34+ cord blood cells in serum-free long-term cultures supplemented with hematopoietic cytokines.

We have previously identified, based on the expression of the CD45RA and CD71 antigens, three major subpopulations of CD34+ cells derived from human umbilical cord blood: CD34+ CD45RAloCD71lo cells (up to 42% multipotent progenitors), CD34+ CD45RA+ CD71lo cells (90% myeloid progenitors), and CD34+ CD45RAloCD71+ cells (70% erythroid progenitors). In the present study, we have investigated the long-term proliferation and differentiation of these subpopulations in response to hematopoietic cytokines. Cells from each subpopulation were cultured for 38 days in serum- and stroma-free liquid cultures supplemented with cytokine combinations that favor either erythropoiesis or myelopoiesis.

The elusive peripheral blood hemopoietic stem cell.

Long-term culture initiating cells (LTC-IC) are primitive hemopoietic progenitors that give rise to clonogenic cells when provided with a supportive feeder layer of mesenchymal cells. These LTC-IC possess many of the characteristics expected of marrow-repopulating "stem cells" including high proliferative and multilineage-differentiative capacity and resistance to 4-hydroperoxy-cyclophosphamide (4-HC) killing. In addition, stem cells are known to persist and may proliferate in murine LTC, and human marrow grown in LTC has been successfully used as hemopoietic support for myeloablative therapy.

Autografting in chronic myeloid leukemia with cultured marrow: update of the Vancouver Study.

When chronic myeloid leukemia (CML) marrow is set up in long-term culture (LTC), Philadelphia chromosome (Ph)-positive (Ph+) cells typically decline and Ph-negative (Ph-) hematopoietic cells often become detectable. In 1987, we initiated a study to evaluate the feasibility of using 10-day cultured marrow autografts to allow intensive treatment of CML. Patients were selected on the basis of a previous assessment of the frequencies of normal and leukemic LTC-initiating cells (LTC-IC) remaining in their marrow after 10 days of LTC.

Maintenance of hematopoiesis in serum-free bone marrow cultures involves sequential recruitment of quiescent progenitors.

We previously described that cells with a CD34+CD71lo phenotype from adult human bone marrow are maintained at constant numbers in long-term suspension cultures supplemented with interleukin-6 (IL-6), IL-3, mast growth factor (MGF) (a c-kit ligand), and erythropoietin (Epo). In view of the large increase in cell numbers in such cultures (for example, > 10(6)-fold per cell), this was an unexpected finding. The following models for the observed maintenance of CD34+CD71lo cells in our cultures were considered: (1) survival of non-dividing cells; (2) self-renewal balanced by loss of cells; (3) asymmetrical divisions; and (4) combinations of the above.

Ontogeny-related changes in proliferative potential of human hematopoietic cells.

Blood cells originate from hematopoietic stem cells that are located at different sites during ontogeny. Production of human stem cells and their progeny in culture is expected to have important implications for experimental therapeutic strategies involving gene transfer and transplantation. Here we report striking differences between primitive hematopoietic cells purified from adult bone marrow, umbilical cord blood, and fetal liver in cytokine-supplemented, serum-free cultures.

Cytokine-induced selective expansion and maturation of erythroid versus myeloid progenitors from purified cord blood precursor cells.

To study the role of different cytokine combinations on the proliferation and differentiation of highly purified primitive progenitor cells, a serum-free liquid culture system was used in combination with phenotypic and functional analysis of the cells produced in culture. CD34+ CD45RAlo CD71lo cells, purified from umbilical cord blood by flow cytometry and cell sorting, were selected for this study because of their high content of clonogenic cells (34%), particularly multipotent progenitors (CFU-MIX, 12% of all cells). Four cytokine combinations were tested: (1) mast cell growth factor (MGF; a c-kit ligand) and interleukin-6 (IL-6); (2) MGF, IL-6, IL-3, and erythropoietin (Epo); (3) MGF, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-3 fusion protein (FP), macrophage colony-stimulating factor (M-CSF), and granulocyte-CSF (G-CSF); and (4) MGF, IL-6, FP, M-CSF, G-CSF, and Epo.

Time lapse video recordings of highly purified human hematopoietic progenitor cells in culture.

Major hurdles in studies of stem cell biology include the low frequency and heterogeneity of human hematopoietic precursor cells in bone marrow and the difficulty of directly studying the effect of various culture conditions and growth factors on such cells. We have adapted the cell analyzer imaging system for monitoring and recording the morphology of limited numbers of cells under various culture conditions. Hematopoietic progenitor cells with a CD34+ CD45RAlo CD71lo phenotype were purified from previously frozen organ donor bone marrow by fluorescence activated cell sorting.

Expression of Thy-1 on human hematopoietic progenitor cells.

Expression of Thy-1 on hematopoietic cells from human fetal liver (FL), cord blood (CB), and bone marrow (BM) was studied with a novel anti-Thy-1 antibody, 5E10. Specificity of 5E10 for human Thy-1 was demonstrated by immunoprecipitation of a 25-35-kD molecule, and the sequence of a cDNA that was cloned by immunoselection of COS cells transfected with a cDNA library derived from a 5E10+ cell line. Two- and three-color immunofluorescence staining experiments revealed that the Thy-1 expression is restricted to, an average, 1-4% of FL, CB, and BM cells, and binding to these cell types is essentially restricted to a very small subset of lymphoid cells and approximately 25% of CD34+ cells.

In vitro properties of purified human stem cell candidates.

Serum-free cultures supplemented with IL-6, IL-3, steel factor, and erythropoietin support extensive production of erythroid cells from purified bone marrow stem cell candidates which are themselves maintained in number in such cultures. In this study, the mechanism responsible for the observed maintenance of primitive hematopoietic cells in rapidly proliferating suspension cultures was examined. The following models were considered: (1) proliferating cells represent a small minority of cells at start of culture (large quiescent pool), (2) self-renewal of primitive cells (balanced by loss through differentiation and death), and (3) asymmetrical divisions (each division of a primitive cell yielding one equally primitive daughter cell and one less primitive, i.

Positive selection of human blood cells using improved high gradient magnetic separation filters.

High gradient magnetic separators (HGMS) create magnetic field gradients that can be used to attract much smaller and less magnetic particles than those required for conventional magnetic separation techniques. As a result cells can be labeled with submicron magnetic particles and still be separated using an HGMS filter. Typically, HGMS filters consist of random arrays of wire such as stainless steel wool.

Phenotypic heterogeneity of primitive leukemic hematopoietic cells in patients with chronic myeloid leukemia.

The peripheral blood of chronic myeloid leukemia (CML) patients with chronic-phase disease and elevated white blood cell (WBC) counts typically contains markedly increased numbers of a variety of neoplastic pluripotent and lineage-restricted hematopoietic progenitors. These include cells detected in standard colony assays as well as their more primitive precursors. The latter are referred to as long-term culture-initiating cells (LTC-IC) because of their ability to generate clonogenic cell progeny detectable after a minimum of 5 weeks incubation on competent fibroblast feeder layers.

Characterization of primitive hematopoietic cells in normal human peripheral blood.

The total number of clonogenic cells present in 5-week-old long-term cultures (LTC) initiated by seeding normal human marrow cells on competent adherent cell feeder layers allows for the quantitation of a more primitive hematopoietic input precursor cell type referred to as an LTC-initiating cell (LTC-IC). Previous studies have suggested that LTC-IC also circulate because production of clonogenic cells continues for many weeks when cells from the light-density (< 1.077 g/mL), T-cell-depleted fraction of normal blood are maintained on irradiated, marrow-derived feeder layers in LTC medium.