Proliferative capacity of murine hematopoietic stem cells in vitro.

Large numbers of granulocytes can be collected repeatedly from the supernatant medium of long-term cultures of mouse bone marrow cells. A constant relationship was found between the number of adherent hematopoietic stem cells and the lifetime cell production per culture. The data indicate that there is a limit to the proliferative capacity of normal and of irradiated stem cells. A similar limitation was found in the production of marked granulocytes from clonal cultures of "beige" C57 (bg/bgJ) stem cells placed in limiting dilutions into stromal culture layers.

Functional organization of the hematopoietic stem cell compartment: implications for cancer and its therapy.

Recent discoveries indicate that hematopoietic stem cells have limits on their proliferative capacity and are unable to divide indefinitely. There is great heterogeneity within the compartment as to the extent of this proliferative limitation. At any given time it appears that hematopoiesis is maintained by the progeny of only a few stem cells. When these are exhausted the progeny from other stem cells take their place. The observations of proliferative limitation, heterogeneity, and clonal succession must be incorporated into any model of stem cell organization. These new discoveries and the models incorporating them have important clinical implications. They may explain the inability of normal tissues to develop drug resistance and they also offer a mechanism by which cell renewal systems decrease the development of malignancies. In the selection of chemotherapeutic agents not only the effectiveness of the drug upon the tumor must be considered, but also how specific agents affect the stem cell compartment. These data have important implications in the use of bone marrow transplantation for both malignant and nonmalignant disease.

Adherent stem cells: frequency in mouse marrow and terminal clone sizes in long-term culture.

Adherent stem cells (ASC) are hematopoietic stem cells of the mouse bone marrow that are equivalent to CFU-S in pluripotency (they produce CFU-S) and proliferative ability (they go through up to 25 doublings). They can be assayed in vitro by a limiting dilution overlay technique in which stromal bone marrow long-term cultures serve as underlayers. In this article we estimate the occurrence rates of ASC in fresh and adherent bone marrow cells and report the distribution of population doublings estimated for 152 ASC clones.

Hemopoietic effects in mice of a transplanted, granulocytosis-inducing tumor.

A transplanted tumor that induces granulocytosis and produces colony stimulating factor (CSF) was studied in mice during several passages. The sequence of events leading to granulocytosis was characterized. Band granulocytes were increased 3 days after tumor inoculation, while simultaneously CFU-s and CFU-c in bone marrow and spleen were transitorily low. This was followed by rapid accumulation of CFU-c and CFU-s in spleen, and by successive waves of increased mitotable and non-mitotable granulocytes in spleen and marrow. In contrast, marrow CFU-c and CFU-s remained normal or slightly decreased.

Cell types associated with fibronectin in long-term mouse bone marrow cultures.

The formation of fibronectin matrix was studied in long-term mouse bone marrow cultures. Stromal and hematopoietic cells were observed in situ under phase contrast optics and quantified according to their staining characteristics on smear preparations. Surface fibronectin was demonstrated by indirect immunofluorescence. While only stromal and no hematopoietic cells participated, various stromal cell types differed in their expression of cell surface fibronectin: Reticulum cells contributed the major portion of fibronectin matrix. Elongated, meshwork-forming histiocytes expressed some surface fibronectin, while the flattened, macrophagic histiocytes remained fibronectin negative. These findings were recapitulated during regeneration of scrape wounds in the adherent layers. Isolated fibronectin matrix did not support hematopoietic cell adherence or maintenance, although it had marked effects on stromal cells.

An in vitro clonal assay of adherent stem cells (ASC) in mouse marrow.

Hematopoietic stem cells with high proliferative capacity can be assayed when stromal bone marrow cultures are overlaid with limiting dilutions of marrow samples. This leads to hematopoietic growth after 4 weeks in a fraction of cultures, consistent with expectations based on Poisson statistics. It will be shown that monoclonal cultures are obtained that last from 2 to 15 weeks and that can generate up to several million mature granulocytes. The originating clone-forming cell is named adherent stem cell (ASC) because of its adherence to plastic or stromal surfaces. The ASC is comparable to the CFU-S in frequency, proliferative capacity and in its ability to give rise to CFU-S. As an unexpected additional finding we report that a mode of "clonal succession" was apparent in cultures which expressed more than one clone.

Hayflick's hypothesis: an approach to in vivo testing.

The experimental evidence relating to the hypothesis of finite cellular life is reviewed. It is emphasized that even if somatic cell production were limited its total potential would have to be vast to provide for extensive cellular regeneration. The actual limit of reproductive cell life would therefore not likely be reached in a normal life-span. It is proposed to test the hypothesis by deliberate exhaustion of stem-cell reserve, and iron-55 cytocide is described as an experimental system that might be applicable.

Forced differentiation of CFU-S by Iron-55 erythrocytocide.

Cascades of Auger electrons are emitted in the decay of 55Fe and absorbed in tissue within a 1 micrometer radius. Cytocidal amounts of 55Fe can therefore eliminate erythroid precursors with minimal damage to adjacent cells. A single intravenous injection leads to continued erythrocytocide in mice because the isotope is reutilized and has a 2.7 year half-life. The cytocide evokes an early compensatory response from morphologically unrecognizable precursors which differentiate into pronormoblasts. These early events leave the granuloid series undisturbed but they are accompanied by a precipitous fall in pluripotent stem cell (CFU-S) numbers in bone marrow, spleen, and blood. The pretreatment levels of CFU-S are not restored. Gradual decline of CFU-S is associated with intermittently increased turnover rates and reduced settings of cell production, yet the capacity for quick restoration of blood loss is unimpaired. The precipitous initial stem cell decrease is not caused by irradiation damage, as shown in a separate experimental series that used the frozen-storage cytocide technique. Only over several weeks could 55Fe radiation accumulate to lethal levels in nondividing stem cells. This irradiation is attributed to incorporation of small amounts of 55Fe into CFU-S, from where it is slowly cleared. The stem cell loss immediately following 55Fe injection is in our interpretation caused by rapid differentiation along the erythroid pathway in a response that involves all progenitor populations. Data are consistent with the hypothesis of limited cell renewal capacity which thereby gains further support.

Residual radiation injury exhibited in long-term bone marrow cultures.

Residual radiation injury was demonstrated in long-term primary cultures of mouse bone marrow. Control cultures underwent three phases of hematopoietic activity as distinguished by initial establishment, steady high (plateau) production of granulocytes, and gradual decline. Irradiation with 50, 300, or 550 rads, given at the end of the initial phase, did not prevent any culture flasks from entering the plateau phase. However, actual production levels and the time they were maintained varied inversely with the radiation dose so that the accumulated postradiation cell production corresponded to an exponential dose-response relationship at any time after treatment. The accumulated cell productions were found to be similar in all groups when expressed by the number of stem cell doublings necessary to produce them. The findings cannot be explained by reproductive cell death and are consistent with the notion of a limited division capacity in hematopoietic stem cells.

Volumes and cellularity in populations of hematopoietic clones.

Volumes of hematopoietic spleen colonies were estimated by measuring parameters of superimposed triaxial ellipsoids. Observations were made on groups of colonies that contained only undifferentiated cells or had differentiated to varying degrees into purely erythropoietic, purely granulocytic or purely megakaryocytic colonies. The average volume occupied by one cell and surrounding matrix, the so-called single cell space, was determined for each type of colony. The estimated colony cellularity was then computed from the measured colony volume and the tabulated single cell space. The techniques of measurement are described. Formulas are provided to estimate mean and variance of volumes and their cell contents. Data on the distribution of spleen colony size and cellularity after seven days of growth are given.

The kinetics of granulopoiesis in long-term mouse bone marrow culture. Part I.

The spontaneous stratification in long-term bone marrow cultures was illustrated and quantified. The cultures were separated into three hematopoietic layers: nonadherent cells in the supernatant medium, lightly adherent cells on top of the stromal layer, and remaining cells buried within the stromal layer. The cells of each layer were subcultured for 10 days in plastic tubes that inhibit the formation of a stromal layer. Daily samplings with absolute and differential cell counts were obtained. We identified three families of cell disappearance curves and cell types: CFU-s, hemocytoblasts, myeloblasts, and promyelocytes (G1, 2); myelocytes (G3); and postmitotic granulocytes (G4). Also, the numbers of mitotic and necrotic cells were determined. The longest half-time of CFU-s was 2.5 days. Lacking stromal support, CFU-s disappeared faster than other differentiated cells. Generally, these cells maintained their numbers for the first week of subcultures, which was attributable to a temporarily maintained balance of cell death and fresh cell production. After more than 7 days, there was a rapid decline of all differentiated cell types.

The kinetics of granulopoiesis in long-term mouse bone marrow culture. Part II.

A mathematical model of mouse granulopoiesis in long-term bone marrow culture was constructed, based on established in vivo cell kinetic parameters. We applied the model to the cell kinetic experiment presented in Part I. Comparing model-predicted cell kinetics with the experimental data led to iterative testing of several hypotheses. In the final model, the cell kinetics of intact tissue culture flasks were reconstructed, using the experimental data from 10 days of tube culture. Among other things, our analysis suggests that the parameters of normal in vivo granulopoiesis apply to bone marrow culture.

The morphology of hematopoietic layers in long-term cultures of mouse bone marrow.

Mouse bone marrow cells in long-term culture were examined with scanning electron microscopy during the first 10 days of growth and with phase contrast microscopy during the first 4 weeks. The development of stroma and hematopoiesis was studied, and phase microscopic observation was used in order to achieve positive cell identifications with scanning EM. We analyzed those cell populations that could not be washed away from the adherent culture layer. These adherent cells in 24-hour cultures contain the full potential of hematopoietic long-term production. Stromal cells started to spread almost immediately and by 5 days had established several layers. Although in early cultures hematopoietic cells were found resting on the surfaces of stromal cells, they were later packed between stromal layers. The blast cells, especially, were usually buried under and between thin sheets of reticulum cells. The study confirms the three-dimensional nature of bone marrow in culture and points to close correspondence with bone marrow structure as studied by others in vivo.

Radiotoxicity of intranuclear 125I atoms not bound to DNA.

The radiotoxicity of 125I covalently bound to DNA is unusually high. This has been attributed both to the Auger electrons which result from the electron capture process accompanying 125I decay and to local transmutation effects which cause extensive damage to nearby structures. We introduced 125I into cell nuclei in the form of iodoantipyrine, a molecule which diffuses freely through cells, and we have compared the survival of these cells to those exposed to radiation from extracellular 125I-labelled albumin or 55Fe-labelled transferrin. We found a value for D0 of 34 rad for 125I decays occurring within the cell nucleus compared to 362 rad for extracellular 125I and 277 rad for extracellular 55Fe. Since transmutation effects are very short range and 125I was distributed uniformly throughout the nucleus rather than bound to DNA, most of the radiotoxicity of intranuclear 125I-labelled iodoantipyrine must be due to Auger electrons.

Selective damage to erythroblasts by 55-Fe.

The low energy and short range of 55-Fe Auger electrons were utilized in mice to deliver lethal intracellular radiation to iron-incorporating erythropoietic precursors with minimal radiation damage to other bone marrow cells. The ensuing intramedullary, selective erythropoietic death was demonstrated by absolute and differential bone marrow cell counts and by decreased blood uptake of 59-Fe. The decreased number of colony-forming units in spleen colony assay and the decreased ability of tranplanted bone marrow to protect fatally irradiated mice shows that the bone marrow was partially depleted of pluripotent stem cells. These data are interpreted to indicate an increased pluripotent stem cell utilization in response to increased demand for differentiation of stem cells along the erythropoietic pathway.

Erythropoietin production in cultures of goat renal glomeruli.

Cultures initiated with isolated goat-kidney glomeruli produce erythropoietin over periods of 7 months. Production seems dependent upon a minimum interval of about 30 days between changes of medium. Experiments distinguishing between erythropoietin, renal activator, and serum substrate support the conclusion that the factor released to the medium of long-term glomerular cultures is erythropoietin itself. The system offers promising opportunities for studying regulation of erythropoietin production, and possibly could be developed as a source of significant quantities of the hormone.

Relevance of specific activity in experimental erythrocytocide by 55Fe.

Iron loads between 0.20 microgram and 26 microgram, added to 5 mu Ci 59Fe, were followed for up to 150 days in mice. Relative organ uptake increased as a function of iron load in liver and kidneys while it decreased in bone marrow and blood. Several weeks after injection, all load-related differences disappeared.

Inappropriate erythropoietin secretion in polycythemia vera.

A patient with classical polycythemia vera (PV) was found to have an inappropriately elevated serum erythropoietin (Ep) level. Investigations did not reveal any lesion or blood abnormality known to be associated with excessive Ep production and erythrocytosis. Sudden withdrawal of blood to reduce the Hb and Hct from 18.5 gm% and 56% to 13.6 gm% and 41.5%, respectively, resulted in an increment of serum Ep to abnormal level. With iron treatment there was a brisk return of Hb and Hct to prebleeding levels which was associated with reduction in the serum Ep. The inverse relationship between the Ep and Hb or Hct is inconsistent with the presence of excessive Ep-producing lesion. These results suggested that the threshold for Ep secretion from normal Ep-secreting tissue to Hb and Hct levels is set at an abnormal level. This patient's marrow cells when cultured in vitro in the absence of Ep, unlike other PV patients' (except one) marrow cells, did not grow erythroid colonies. In the presence of Ep, however, the colonies comparable to those formed from normal marrow cultures were obtained. These results suggested that his marrow erythropoietic cells were neither Ep independent nor Ep-hyperresponsive, as has been suggested by some investigators for erythropoiesis in PV. This patient presents phenomena that hitherto have not been reported.