Documentation of normal and leukemic myelopoietic progenitor cells with high-resolution phase-contrast time-lapse cinematography.

The high-resolution phase-contrast, time-lapse cinematography using oil immersion lenses and 16-mm film demonstrates the kinetic cell events as maturation, locomotion, mitosis, and apoptosis of cells cultivated at 37 degrees C for up to 10 days. 0.5 v/v frozen-thawed sera with presumably high cytokine concentrations were added to the plasma or agar clot. Vital progenitor cells from human bone marrow and blood have a large, bright, unstructured nucleus with a large nucleolus and a narrow rim of cytoplasm (nuclear/cytoplasmic volume ratio = 0.7). Their nuclei are 6-14 micrometer in diameter and double their volume within 8 h. Many (70%) move at a mean speed of 2 micrometer/min, and many (30%) multiply with alpha-2alpha mitoses, generating progenitor cell families. Various disturbances during the course of mitosis lead to the formation of polyploid cells, thereby yielding the megakaryocytic cell line. Some of the progenitor cells undergo asymmetric alpha-alphan mitoses: One of the two initially identical daughter cells remains a progenitor cell in the morphological sense, whereas the other daughter cell - depending on the size of its mother cell - matures in the same culture medium to form a granulocytopoietic, monocytopoietic or erythrocytopoietic cell line. - In acute myeloid leukemias (AML), the blasts and their nuclei are slightly larger than the corresponding progenitor cells and move faster (5 micrometer/min). Symmetric alpha-2alpha mitoses permit unlimited multiplication of the leukemic blasts if contact with cytotoxic lymphocytes does not render them apoptotic. This results in more stromal cells than normal. Granulocytopenia, monocytopenia, and anemia occur due to the genetic impairment of signaling control for asymmetric alpha-alphan mitoses, and thrombocytopenia occurs due to the reduction in polyploidization.

Human megakaryoblastic proliferation and differentiation events observed by phase-contrast cinematography.

Microcinematographic documentation of mitoses, amitoses, endomitoses, or cytoplasmic fusion shortly after completion of mitoses was done in bone marrow specimens of patients with quantitative platelet disorders and controls. In patients with platelet disorders, most mitoses with cell duplication occurred in large promegakaryocytes after 4-fold nuclear and cytoplasmic enhancement. Normal specimens showed polyploidization happening in small megakaryoblasts, while mitoses with cell duplication were seen only after cultivation in freeze-thawed sera of patients with platelet disorders. Frequently, lymphocytes were observed to contact megakaryoblastic cells undergoing mitoses, amitoses and endomitoses and to enter the cytoplasm of megakaryocytes (emperipolesis), leaving it again after several hours.

Locomotion of human bone marrow and peripheral blood leukocytes is associated with maturational stage and altered in malignancy.

Using high-resolution phase contrast time-lapse microcinematography, slow movements (0.8-2.0 microns/minute) of human myeloblasts, monoblasts, and megakaryocytes can be recorded. Upon maturation to promyelocytes, motility is lost until cells have reached the stage of metamyelocytes (0.4 microns/minute). Motility increases sharply following maturation into segmented neutrophils (20.4 microns/minute). Monocytes and promonocytes display a mean track velocity of 7.1 microns/minute. The distribution of lymphocyte velocities is not bell-shaped but shows three maxima of 2.1 microns/minute, 7.8 microns/minute, and 18.4 microns/minute. Atypical lymphocytes from patients with infectious mononucleosis belong to the fast group, whereas lymphocytes activated in vitro by mitogens belong to the slow group. Red blood cell precursors from normal human bone marrow do not move actively. In contrast, erythroleukemic blasts show a motility comparable to normal myeloblasts. Similarly, acute promyelocytic leukemia cells move at 6.7 microns/minute, while their normal counterparts are sessile. Increased motility is also observed in blast cells from a variety of acute myelogenous and lymphoblastic leukemias.

Measurements of nuclear sizes of committed hematopoietic progenitor cells compared to leukemic blasts from human bone marrow smears.

By measurements of the nuclear sizes of normal and of leukemic myeloblasts and monoblasts in colored smears, the differences estimated in surviving cells with phase contrast were confirmed. The median nuclear size of normal myeloblasts is the smallest one and differs significantly from that of the normal monoblasts and AML myeloblasts. The median nuclear size of normal monoblasts significantly exceeds values of normal myeloblasts and of AMoL monoblasts. Both leukemic blasts cover a wide range of nuclear diameters, but do not differ from one another, while normal myeloblasts have a smaller dispersion than normal monoblasts. The difference of leukemic blasts and normal progenitor cells is to be emphasized.

Lymphocytes as inducers of mitoses of human morphologically identifiable progenitor cells. Phase contrast observations.

Phase contrast time lapse observations of human bone marrow cells demonstrate the induction of progenitor cell mitoses or amitoses by the touch of locomotive lymphocytes on the cell surface. Interkinetic progenitor cells as well as precursor cells before mitosis were very rarely touched by lymphocytes. We suppose this cell-cell interaction is essential in the T-cell participation in hematopoietic stem cell proliferation.

Origin of the early megakaryopoietic progenitor cell and further polyploidization in the thrombopoietic cell series. Phase-contrast observations of human bone marrow.

Continuous observation of living cells by phase-contrast time lapse microcinematography revealed different origins of multinuclear cells and cells with lobed nuclei in the hematopoietic progenitor cell series, which must be classified as megakaryoblasts, the determined progenitor cells of the megakaryopoietic series. Hematopoietic progenitor cells with single nuclei of nuclear diameter 5-13 microns, the promegakaryoblasts, may become polyploid by various means: by postmitotic cytoplasmic fusion, by consecutive nuclear fusion, by endomitosis, or by nuclear amitosis or lobe formation presumably after endoreduplication and nuclear growth. Including amitotic nuclear division in the mitotic rate of normal progenitor cells nearly the half of all these cells in G2 phase (0.4) produce in vitro octoploid megakaryoblasts, respectively osteoclasts. Thrombopoietin-rich sera in the medium enhance the mitotic index of the hematopoietic progenitor cell line from 0.1 to 0.25, but do not change the proportion of polyploid mitoses.

Evidence for a rat granulocyte chalone effect on the proliferation of normal human bone marrow and of myeloid leukemias.

Evidence is presented that rat ascites cell extracts, acting as granulocyte chalone, temporarily inhibit the cell doubling of granulocytopoietic precursors in suspension cultures of human bone marrow. The extracts do not affect the proliferation of erythroblasts and do not show any cytotoxicity. In addition, a relative increase of granulocytic precursors capable of proliferation was found, suggesting an increase in the resting population of the granulocytopoietic proliferation pool due to extract treatment. However, the extract capable of depressing the proliferation of the normal granulocytopoiesis did not affect the granulocytopoiesis of 7 chronic myeloid and 2 chronic myelomonocytic leukemias at the same dose level. In contrast to these chronic leukemias, blast proliferation of 4 acute myeloid and monocytic leukemias was greatly depressed not only for 6 h but for the whole culture period of 48 h.

Maturation and proliferation capacity of blood cells from untreated acute myeloid leukemia and its prognostic significance.

Blood cells from 68 patients with untreated acute myeloid luekemia were cultured in RPMI-medium without stimulating factors up to ten days. The cultures showed in part maturation and proliferation to monocytes-macrophages, in part to promyelocytes, myelocytes and Pelger-like cells, in part we did not find any differentiation or the cultures were degenerated during the first days. Retrospectively we found that in the 16 blood cell cultures with capacity to differentiation into the monocyte-macrophages-system 5 patients had a smouldering leukemia. Our preliminary evidences suggest that the diagnosis "smouldering leukemia" is to be found with out in vitro culture system. Further analysis suggest that patients with acute leukemia whose blood cells have the capacity for maturation to monocytes-macrophages or to promyelocytes, myelocytes and Pelger-like cells have a better chance of achieving a complete remission and a longer median survival time.