Optimizing limiting dilution assays: frequency and 'ability' measurements of haemopoietic progenitor cells.
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Cell Count↗
Biomedical subjects
M Y Gordon
Publications and source records attributed to M Y Gordon.
Phenotype and progeny of primitive adherent human hematopoietic progenitors.
Hematopoietic progenitor cells can be classified as plastic- and stroma-adherent (P+S+), stroma-adherent (P-S+) and non-adherent (P-S-). Both P+S+ and P-S+ populations are detected in delta (delta) culture systems where they produce non-adherent (P-S-) granulocyte-macrophage colony-forming cells (CFU-GM) and erythroid burst-forming units (BFU-E). Here we demonstrate that the plastic-adherent progenitor cells (P delta cells) comprise 5-10 percent of the CD34+, population in adult human marrow. Moreover, they do not express CD3 or CD22 and 88 percent of them are CD38-, 88 percent are CD33- and 74 percent are HLA-DR-. Production of CFU-GM by purified plastic-adherent CD34+, adherent cells was 60 percent of the number produced by recombined CD34+, and CD34- fractions. We have shown also that the plastic-adherent P+S+ cells are the precursors of the stroma-adherent P-S+ cells (S delta cells), day 21 cobblestone-area forming cells (CAFC) and cells capable of sustained hematopoiesis in a modified long-term bone marrow culture system. These observations support the primitive nature of P delta cells and establish a phenotypic sequence of plastic and stroma adherence through stroma adherence to non-adherence in hematopoietic cell development. To further investigate the relationship between P delta cells, S delta cells and long-term culture-initiating cells (LTCIC), we cultured whole mononuclear cell tractions and plastic-adherent cell-depleted mononuclear cell fractions in long-term culture and in the S delta assay. The results indicated the P delta cells were inhibited in the presence of stromal cells.
ADP-ribosyl Cyclase↗
Cellular and molecular mechanisms in chronic myeloid leukaemia: biology and treatment.
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Humans↗
Plastic-adherent progenitor cells in mobilized peripheral blood progenitor cell collections.
The use of peripheral blood progenitor cells (PBPC) to reconstitute hematopoiesis after high-dose chemoradiotherapy is now commonplace in the treatment of malignancies. Attempts to characterize these cells have concentrated primarily on their phenotype and their content of clonogenic colony-forming cells (CFC). We have used a plastic-adherent delta (P delta) assay system to evaluate the quantity and quality of more primitive cells in addition to the conventional measurements of CFC and CD34-positive cells. The leukapheresis products from 20 patients mobilized using cyclophosphamide (Cy) and granulocyte colony-stimulating factor (G-CSF) were examined for progenitor cell content. The mean number of mononuclear cells (MNC), colony-forming units-granulocyte/macrophage (CFU-GM), and CD34-positive cells from two leukaphereses per patients were 7.9 x 10(8)/kg, 47.3 x 10(4)/kg, and 10.5 x 10(6)/kg, respectively. The mean number of P delta progenitors was 9.3 x 10(4)/kg. Limiting dilution analyses showed the frequency of P delta progenitors in PBPC to be between 1 and 5.3 per 10(5) MNC and that each P delta progenitor has the proliferative capability to generate an overall mean of 4.5 CFU-GM. Of the 20 patients, 16 underwent autografting with PBPC alone. Fifteen patients engrafted neutrophils and platelets within 16 days. One patient had delayed engraftment associated with inadequate etoposide clearance. Statistical analysis showed a strong correlation between numbers of CFU-GM and CD34 positivity. The numbers of plastic-adherent P delta progenitor cells did not correlate with CFU-GM or CD34-positive cells. We conclude that the plastic-adherent P delta progenitor cell assay is capable of measuring primitive hematopoietic cells and that it may be useful for the investigation of primitive progenitors in PBPC harvests.
Antigens, CD34↗
Interaction of interleukin 7 (IL-7) with glycosaminoglycans and its biological relevance.
We have investigated the binding of interleukin 7 (IL-7) to sulfated glycosaminoglycans and evaluated its biological consequences. IL-7 binds to heparin and heparan sulfate, to a lesser extent to dermatan sulfate and does not bind to chondroitin sulfate. It was eluted from heparin by 0.3-0.6 M NaCl and from heparan sulfate by < 0.3 M NaCl. We also measured the affinity of IL-7 for heparin using an affinity co-electrophoresis method and found an affinity of 25 nM. In spite of these findings, IL-7 does not bind to the S17 cell line which supports lymphopoiesis. However, addition of heparin to cultures of an IL-7-dependent pre-B cell line (2E8) inhibited IL-7-stimulated proliferation and IL-7 complexed with heparin was more resistant than free IL-7 to protease treatment. Taken together, these results suggest that heparin may act as a carrier for IL-7, blocking its interaction with target cells and protecting it from degradation during transit.
Animals↗
Progenitor cells in the blood and marrow of patients with chronic phase chronic myeloid leukaemia respond differently to macrophage inflammatory protein-1 alpha.
Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is a negative regulator of normal haemopoietic stem cell proliferation. Insensitivity to MIP-1 alpha of progenitor cells in chronic myeloid leukaemia (CML) could, therefore, explain myeloid expansion in this disease. We compared the effects of MIP-1 alpha on progenitor cells in normal marrow and in the blood and marrow of patients with chronic phase CML. Plastic-adherent precursors of granulocyte-macrophage colony-forming cells (P delta progenitors) are very primitive progenitor cells and are detected by incubating them for 1 week in liquid culture and assaying the CFU-GM released into the supernatant. Direct CFU-GM assays were also used in this study. Daily addition of 300 ng/ml/day of MIP-1 alpha to P delta progenitor assays of normal marrow cells suppressed CFU-GM production by 50% and in CML bone marrow P delta cultures by 20-30%. The response of CML blood P delta progenitors was heterogeneous. In five of nine cases, CFU-GM production was doubled in the presence of MIP-1 alpha and in four of nine cases, it was reduced. Addition of 100-500 ng MIP-1 alpha to direct assays of CFU-GM stimulated colony formation by normal marrow and CML blood cells to a similar extent.
Bone Marrow Cells↗
In search of the haemopoietic stem cell.
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Bone Marrow Transplantation↗
Probability of progenitor renewal (PPR) and production of clonogenic progeny (CFU-GM) by primitive adherent progenitor cells in adult human bone marrow and umbilical cord blood.
Human haemopoietic tissues contain primitive plastic-adherent progenitor cells (P delta cells) that can be detected by measurement of their granulocyte-macrophage colony-forming cell (CFU-GM) progeny. Limiting dilution analysis and Poisson statistics are necessary for determining the frequency of P delta cells because each of them produces several CFU-GM. Limiting dilution also permits measurement of the abilities of individual P delta progenitors to produce CFU-GM. Here we report that the frequencies of P delta progenitors in cord blood and adult marrow are similar (5.6 and 7.8/10(5) mononuclear cells respectively) and individual cord blood P delta progenitors produce fewer CFU-GM than adult P delta progenitors. To test the possibility that the lower production of differentiated progeny by cord blood cells was the result of a higher rate of self-renewal, we devised a two-stage limiting dilution assay relying on the relative production of CFU-GM after two consecutive weeks of incubation. The probability of progenitor renewal (PPR) was derived from the number of wells (progenitors) that produced CFU-GM on both occasions compared with the number that produced CFU-GM on the first occasion only. The total number of CFU-GM produced on the second occasion compared with the number produced on the first occasion provided an index of the overall change in the size of the P delta cell population. The data indicate that P delta cells in cord blood have a higher PPR (0.59) than those in adult marrow (0.36). Also, the relative numbers of CFU-GM produced in the second and first weeks were greater for cord blood (1.2) than for adult marrow (0.36). Therefore P delta cells in cord blood have a greater capacity for self-maintenance and possibly for expansion than P delta cells in adult marrow.
Adult↗
Apoptosis in chronic myeloid leukaemia: normal responses by progenitor cells to growth factor deprivation, X-irradiation and glucocorticoids.
Inhibition of apoptosis (genetically programmed active cell death) by p210 BCR-ABL expression is a mechanism that might contribute to clonal expansion in chronic myeloid leukaemia (CML). Since cell death following exposure to ionizing radiation and many chemotherapeutic agents can occur by the apoptotic pathway, inhibition of apoptosis would be expected to confer a relative resistance to these treatments. Similarly, cells deprived of growth factors in vitro die by apoptosis, and inhibition of apoptosis would therefore be expected to allow cells to survive better in growth factor-deprived conditions. We found that the survival of normal and CML myeloid progenitors was the same after in vitro incubation in deprived conditions and after treatment with X-irradiation or glucocorticoids. We also found that mature cells in colonies produced by CML progenitors (CFU-GM) did not survive better than those produced by normal progenitor cells. Flow cytometric analysis of propidium iodide-stained cells provided a direct indication that the degree of apoptosis may correspond to the degree of deprivation. These results suggest that inhibition of apoptosis may not be the primary mechanism whereby BCR-ABL influences the expansion of the malignant clone in CML.
Apoptosis↗
Evidence for a mechanism that can provide both short-term and long-term haemopoietic repopulation by a seemingly uniform population of primitive human haemopoietic precursor cells.
One of the controversies surrounding the repopulating capacities of haemopoietic stem cells is whether or not the same or different populations are responsible for short-term and long-term repopulation after transplantation. To address this question, we analysed results obtained from an in vitro model for the clonal production of granulocyte-macrophage colony-forming cells (CFU-GM) by individual primitive multilineage precursors in adult human bone marrow. The primitive precursors adhere to plastic and produce CFU-GM in a 1-week long 'delta' type culture. The clones that form are classified as having short maturation pathways (clones containing predominantly day 7 CFU-GM) or long maturation pathways (clones containing predominantly day 21 CFU-GM). The results indicate that individual primitive (P delta) cells produce clones that reach full maturity after different periods of time so that cells corresponding to a range of maturational stages can become available simultaneously. Consequently, transplanted stem cells may be able to provide both rapid and long-term mature cell recovery whilst at the same time reconstituting the stem cell pool. These results suggest that it might be possible to use highly purified stem cell populations, devoid of committed progenitors, for clinical transplantation.
Bone Marrow Cells↗
Some factors determining the minimum number of cells required for successful clinical engraftment.
Theoretically, a single pluripotent haemopoietic stem cell should be able to reconstitute haemopoiesis following transplantation. However, clinical and experimental observations demonstrate that it is necessary to transplant numerous stem cells to obtain engraftment. Here we discuss some of the reasons for this apparent discrepancy and provide some quantitative estimates of the influence of kinetic factors in determining the number of stem cells required for clinical engraftment.
Cell Count↗
Extent of variability inherent in measurements of CD34-positive cells in different human haemopoietic tissues.
Estimation of CD34 expression is widely used to detect and quantify progenitor cells in haemopoietic tissues used as stem cell sources for transplantation. Mouse monoclonal antibodies to CD34 recognise different epitopes of the mucin-like sialoglycoprotein. These epitopes can be grouped into three classes by their differing sensitivities to the enzymes: neuraminidase, chymopapain and glycoprotease. We have compared the expression, by flow cytometry, of the three CD34 epitopes on normal adult and fetal haemopoietic tissue and in chronic myeloid leukaemia, and have used four antibodies from each class to assess variability of staining within and between epitope classes. The results reveal variable expression of CD34 both within and between tissue types and antibody classes. As a result of the different levels of detection by different antibodies, the apparent number of CD34-positive cells vary by approximately 6-fold. Enrichment for CD34 cells using magnetic bead technology shows a significant difference in the percentage of CD34 cells detected for two of the epitope types.
Adult↗
Expression of cell-surface lectins on haemopoietic progenitor cells.
The expression of lectins on the surface of the murine multi-myeloid progenitor cell line FDCP-Mix, and the human leukaemic cell line KG1, was assessed and compared to the pattern of lectin expression observed on human bone marrow mononuclear cells. Using flow cytometry, cell-surface lectins were identified by their ability to recognise fluorescein isothiocyanate-labelled neoglycoproteins. Both cell lines recognised neoglycoproteins expressing alpha-D-glucose and alpha-D-galactose residues. Inhibition studies suggested that recognition of these neoglycoproteins was via two independent receptors, each displaying characteristic sugar-binding properties. The CD34+ population of bone marrow mononuclear cells, identified by positive staining with the anti-CD34 antibody QBend10, were shown to interact with alpha-glucose-, alpha-galactose- and alpha-D-mannose-expressing neoglycoproteins. Similarly, binding of these probes to lymphocyte and monocyte sub-populations of CD34- bone marrow mononuclear cells was observed. In contrast, CD34- granulocytic cells did not appear to recognise these probes. It is suggested that the alpha-D-galactose binding activity observed for both cell lines and the alpha-D-galactose and alpha-D-mannose binding activity observed for bone marrow mononuclear cells represent expression of the component polypeptides of the previously reported galactosyl/mannosyl receptor. The glucosyl-specific receptor, observed on both cell lines and on bone marrow mononuclear cells, has not been reported previously. It is suggested that this receptor may mediate glucose transport or cell adhesion through recognition of glucosyl-containing compounds such as heparan sulphate.
Bone Marrow Cells↗
Stochastic effects in hemopoiesis.
Stochastic effects underlie all hemopoietic cell responses but can only be observed at the level of one or a very few cells. Here we have considered the relevance of stochastic effects to aspects of hemopoietic cell development.
Animals↗
Stem cells and the microenvironment in aplastic anaemia.
Normal blast colony-forming cells (BI-CFC) bind to stroma cultured in the presence of methylprednisolone (MP+) but not to MP- stroma. In aplastic marrow, the incidence of BI-CFC is variable (0-4 x normal values) and there is no consistent relationship with the CFU-GM (granulocyte-macrophage colony-forming cell) content. Normal stroma require MP to induce BI-CFC binding function and form fat cells whereas MP- stroma grown from 4/9 aplastic patients formed fat cells and bound BI-CFC. The 5/9 aplastic cases that did not form fat cells spontaneously also bound BI-CFC moderately better than normal stroma. This suggests that the haemopoietic microenvironment in aplastic anaemia responds physiologically to bone marrow failure by increasing its haemopoietic support capacity.
Adipocytes↗
Physiology and function of the haemopoietic microenvironment.
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Anemia, Aplastic↗
SLe(x) expression of normal CD34 positive bone marrow haemopoietic progenitor cells.
We investigated sialylated Lewis x (sLe(x)) antigen expression on CD34 positive (CD34+) haemopoietic progenitors in the bone marrow of eight healthy volunteers using monoclonal antibodies. We found that in all the samples examined, CD34+ bone marrow progenitors strongly expressed the sLe(x) antigen. This contradicts previous publications which reported sLe(x) expression on malignant blast cells but not on normal CD34+ progenitor cells.
Antigens, CD↗
The kinetics of colony formation by CFU-GM in vitro.
Video-recordings of whole normal bone marrow granulocyte-macrophage colony (CFU-GM) cultures were made after 7, 14 and 21 d. Retrospective viewing of the tapes allowed the relationships to each other of the colonies scored on the three different occasions to be documented. The results show that, according to our scoring criteria, there is very little overlap between the numbers of colonies scored on days 7, 14 and 21. Moreover, only about 10% of the progenitors in a sample form day 21 colonies. The remaining progenitors form colonies earlier in the culture period and either disappear before day 21 or remain small.
Bone Marrow Cells↗