Reconstruction of the hematopoietic system after stem cell transplantation.

The practice of hematopoietic stem cell transplantation to rescue patients from the myeloablative effects of chemo- or radiotherapy, or to replace defective hematopoiesis, is based on the assumption that hematopoietic stem cells in the graft have sufficient proliferative potential to supply mature blood cells for the remainder of the recipient's lifespan. However, the mechanism(s) whereby this is achieved are not well understood. Here we address the reconstruction of the hematopoietic system by considering the effects of stem cell and progenitor cell renewal and differentiation. We conclude that stem cell self-renewal is necessary for hematological recovery and that infused committed progenitor cells (CFU-GM) may contribute to the neutrophil count in the early posttransplant period.

Interleukin 3 (IL-3), but not stem cell factor (SCF) increases self-renewal by human erythroid burst-forming units (BFU-E) in vitro.

Interleukin 3 (IL-3) and stem cell factor (SCF) are both important regulators of early haemopoietic cell development. Here, we have compared their effects or the kinetics of erythroid burst formation by BFU-E in normal adult bone marrow. We grew the BFU-E in the presence of erythropoietin (Epo) alone, Epo + IL-3 or Epo + SCF and scored the numbers of subcolonies in individual bursts after 14 days. The data were plotted as the cumulative distribution of the numbers of subcolonies per erythroid burst then linearised by logarithmic transformation. Analysis of the data revealed that IL-3 increases the numbers of subcolonies in BFU-E whilst SCF increases the size of the subcolonies themselves. Experiments involving combinations of Epo + IL-3 + SCF and the delayed addition of IL-3 or SCF indicated that the actions of IL-3 and SCF are largely independent of one another. We conclude that: (1) IL-3 acts at an earlier stage of erythroid differentiation than SCF, and (2) it may be possible to classify haemopoietic growth factors according to their effects on cell kinetics in vitro.

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.

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.

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.

Routes to repopulation--a unification of the stochastic model and separation of stem-cell subpopulations.

In summary, we propose that the haemopoietic system consists of a hierarchy of clonogenic stem and progenitor cells and that the stochastic effect is superimposed upon these compartments. This means that (i) considerable heterogeneity can exist within major compartments without the need to invoke the existence of numerous subpopulations, and (ii) that the stochastic effect and the separation of stem cells are not totally incompatible. The unifying model offers a reasonable explanation of the situations summarized above. The clear implication of the disappearance of stem cell clones as a result of the stochastic effect is a requirement for multiple stem cells to ensure long-term repopulation in cell replacement or gene therapy. The necessary multiplicity may not be great, possibly 3-fold according to the results of Lemishka, and the scheme shown in Figure 1, but should be considered in the design and interpretation of experimental and therapeutic strategies. Other implications are concerned with the degree to which it is necessary to select 'subpopulations' of haemopoietic stem cells for transplantation or experimental work.

The kinetics of cell cloning assays: hemopoietic spleen colony growth.

The less than self-evident effect of alterations in cell kinetics on the growth of hemopoietic colonies in the spleens of irradiated mice has important implications for the interpretation of results obtained with this unique multipotential stem cell assay. The effect on colony growth of variation in the principle cell kinetic parameters has been calculated by simulating the growth of spleen colonies so as to provide a guide to the interpretation of experimental results. This data is used to re-interpret two particular experimental observations on the effect of cytotoxic agents in terms of alterations in the response of stem cells to regulatory factors in place of previous interpretations regarding the differing self-renewal capacities for subpopulations of stem cells and their sensitivity to various drugs.

Haemopoietic spleen colony growth: a versatile, parsimonious, predictive model.

Substantial support has been obtained for the stochastic model for stem cell differentiation first proposed by Till, McCulloch & Siminovitch (1964), over 20 years ago. By adding a cell maturation pathway, it is possible to predict (by computer simulation) the total number of cells and consequently the time at which individual colonies appear and disappear. Only a few uncontroversial assumptions are required to predict that cells, uniform with respect to self-renewal, are capable of producing the high proportions of late disappearing and late appearing colonies observed experimentally in the spleens of irradiated mice that have been injected with normal haemopoietic cells. It is shown that differences in stem cell self-renewal only slightly influence the time of appearance of colonies; whereas changes in the kinetics of the maturing cells, by changing the size of colonies, has a marked effect on the time of appearance and disappearance of colonies and on the average doubling-time of colony-forming cells per colony (but not the doubling-time of individual colonies). These results (1) seriously question the prevailing view that spleen colonies scored at 8 days measure a separate population (without the capacity for self-renewal), from those scored at 12 days; (2) argue against the existence of multiple sub-populations of stem cells with differing self-renewal and toxicity to cytotoxic agents; (3) help to identify those experiments for which it is obligatory to postulate heterogeneity, and (4) are consistent with self-renewal being regulated by a feedback control of stem cell differentiation, to which only proliferating stem cells can respond and where the stimulus for differentiation decreases at a time when the bone marrow is known to be depleted.

A new look at the haemopoietic response to cytotoxic agents and stem cell regulation.

A re-evaluation of the response of haemopoietic stem cells, in mice, to radiation and anticancer drugs suggests a precise mechanism for the regulation of the number of stem cells through a control of their rate of differentiation. This analysis which has some counter-intuitive aspects, appears to be the first attempt to obtain a unified explanation for various effects of cytotoxic agents and has some important biological and clinical implications.

The accumulation of an aryloxylkylamidine (501C) and 5-hydroxytryptamine in human polymorphonuclear leucocytes: a quantitative electron microscopy study.

The uptake of anilino-N-2-m-chlorophenoxypropylacetamidine (501C) and 5-hydroxytryptamine (5-HT), in human polymorphonuclear leucocytes (PMN) was investigated by quantitative ultrastructural autoradiography. The major concentration of both drugs was in the cytoplasmic granules, 71.5 +/- 6.8% for [3H] 501C, and 68.75 +/- 6.1% for [3H]5-HT. Lesser quantities of both drugs were associated with the nucleus; 18.1 +/- 4.8% for [3H] 501C and 23.0 +/- 4.2% for [3H] 5-HT. Only small amounts of activity were recorded at other sites. The data suggests that there may be common binding sites for 501C and 5-HT in PMN. Furthermore the concentration of 501C in PMN granules may also account for the damaging effect of high concentrations of the drug on PMN.

The interaction of 125I-insulin with cultured 3T3-L1 adipocytes: quantitative analysis by the hypothetical grain method.

The murine 3T3-L1 fibroblast under appropriate incubation conditions differentiates into an adipocyte phenotype. This 3T3-L1 adipocyte exhibits many of the morphologic, biochemical, and insulin-responsive features of the normal rodent adipocyte. Using quantitative electron microscopic (EM) autoradiography we find that, when 125I-insulin is incubated with 3T3-L1 adipocytes, the ligand at early times of incubation localizes to the plasma membrane of the cell preferentially to microvilli and coated pits. When the incubation is continued at 37 degrees C, 125I-insulin is internalized by the cells and preferential binding to the villous surface is lost. With the internalization of the ligand, two intracellular structures become labeled, as determined by the method of hypothetical grain analysis. These include large clear, presumably endocytotic, vesicles and multivesicular bodies. Over the first hour of incubation the labeling of these structures increases in parallel, but in the second hour they diverge: the labeling of multivesicular bodies and other lysosomal forms continuing to increase and the labeling of large clear vesicles decreasing. At 3 hours limited but significant labeling occurs in small Golgi-related vesicles that have the typical distribution of GERL. The distinct morphologic features of this cell make it ideal for a quantitative morphologic analysis and allow for an unambiguous view of the sequence of events involved in receptor-mediated endocytosis of a polypeptide hormone. These events are likely to be representative of the processing of insulin by the mature rodent adipocyte.

Ultrastructural studies on the thymus gland after the administration of phenylhydrazine to bank voles (Clethrionomys glareolus).

The thymus glands of wild and laboratory reared bank voles (Clethrionomys glareolus) were examined for ultrastructural changes before and after the administration of low (20 mg/kg) and high (60 mg/kg) doses of phenylhydrazine (PhZ) given intraperitoneally on days 0, 1 and 3 of a dosing regime. Erythrocytes developed in focal aggregations in the subcapsular and outer cortical zones. There was a marked depletion of the subcapsular cortex following drug treatment that was reversed with time. A large number of granulocytes were visible in the cortex after PhZ, and mast cells developed in situ.

Receptor-mediated endocytosis of insulin: role of microvilli, coated pits, and coated vesicles.

When 125I-labeled insulin (125I-insulin) is incubated with 3T3-L1 adipocytes and cells processed for electron microscopic autoradiography, the ligand initially localizes preferentially to microvilli and coated pits. As a function of time and temperature, this initial preferential localization to microvilli is lost, and the ligand is internalized by the cell. Serial sections of apparent coated vesicles near the cell surface indicate that about half of these structures are true vesicles and, therefore, intermediates in this receptor-mediated endocytotic process. With time, 125I-insulin localizes to larger intracellular membrane-bounded structures. When cells are incubated with another ligand, cationic ferritin, that is taken up by adsorptive endocytosis, essentially the same structures are involved as for the endocytosis of 125I-insulin. The data suggest that specificity for receptor-mediated endocytosis is conferred by the specific ligand receptor and possibly by ligand-induced receptor mobility in the plane of the plasma membrane. Other structures such as coated pits, coated vesicles, larger vesicles, and secondary lysosomes are common for different ligands.

A regulatory mechanism for the number of pluripotential haemopoietic progenitor cells in mice.

Large changes in the pattern of regeneration of haemopoietic progenitor cells (CFU-S) are reported as a result of simply altering the time interval between administration of certain cytotoxic agents and whole-body irradiation. These results suggest a negative feedback control for the number of CFU-S, which has an appreciable time delay. A high inverse correlation between the number of maturing granulocytic cells (with ring-shaped nuclei) at the time of irradiation and the subsequent regeneration of CFU-S is consistent with these cells producing some substance that controls the number but not the proliferation rate of the CFU-S. The possibility is considered that this mechanism is an important control in the biological regulation of haemopoiesis.

Radiosensitivity and recovery of two murine haemopoietic progenitor cell populations following gamma rays and neutrons.

Experiments measuring the sensitivity of mouse bone marrow stem cells (CFU-S) and granulopoietic progenitor cells (CFU-C) to gamma-rays and neutrons are described. Both populations are more sensitive to neutrons than to gamma-rays, and in each case CFU-S are more sensitive than CFU-C. The CFU-C (but not CFU-S) show a biphasic dose-response curve to gamma-rays, and the data suggest that 20-50% of CFU-C in normal mice are more resistant to gamma-rays than the rest, possibly due to hypoxia. This difference between the two cell populations may be related to differences in spatial location in the bone marrow. The recovery of these cells following gamma-rays is consistent with previous data which suggests that CFU-C are not self-maintaining, but require to be fed in from the stem cells.