J. L. Chertkov

Lifelong hematopoiesis in both reconstituted and sublethally irradiated mice is provided by multiple sequentially recruited stem cells

OBJECTIVE To evaluate the dynamics of stem cell production to hematopoiesis, the number of active stem cell clones and the lifespan of individual clones were studied. MATERIALS AND METHODS The clonal contribution of primitive hematopoietic stem cells (HSC) responsible for long-term hematopoiesis was determined using two approaches. In one model, irradiated female mice were reconstituted with retrovirally marked male hematopoietic cells. In the second model, mice were irradiated sublethally without hematopoietic cell transplantation. In both models, bone marrow cells were serially sampled from the same mouse throughout a 12- to 20-month period and injected into irradiated recipients for analysis of day 10 colony-forming unit-spleen (CFU-S). The donor origin of CFU-S was determined by the presence of retrovirally marked cells or cells with chromosomal aberrations. RESULTS The results of the two essentially different models show that 1) hematopoiesis is mainly the product of small clones of hematopoietic cells; 2) the lifespan of the majority of clones is only 1 to 2 months; 3) the clones usually function locally; and 4) the vast majority of the clones replace one another sequentially. Primitive HSCs capable of producing long-lived clones (about 10% among all clones), which exist during the entire life of a mouse, were detected by the radiation-marker technique only. CONCLUSION Multiple short-living clones (at least on the level of CFU-S production) comprise the vast majority of the active stem cells in transplanted recipients or after endogenous recovery from sublethal irradiation.

Cells responsible for restoration of haemopoiesis in long-term murine bone marrow culture.

Long-term haemopoiesis in bone marrow culture is sustained by the progeny of haemopoietic progenitor cells (HPC), which differ from CFUs by very low sensitivity to repeated hydroxyurea (HU) injections. The transit time of a haemopoietic clone from HPC to cells proliferating in culture is 6-7 weeks. The results suggest that the stem cell continuum is an expansion type compartment, members of which gradually lose their proliferative potential during differentiation.

Cells forming spleen colonies at 7 or 11 days after injection have different proliferation rates.

Abstract In the early periods (7–9 days) after haemopoietic cell injection, colonies produced by CFU‐s and by their progeny are identified in the spleen, while at later periods (11 days after injection) only spleen nodules produced by CFU‐s persist. the increase in the suicide values of CFU‐s after sublethal (2 Gy) irradiation of mice is associated with a higher proliferation rate of precursors of transitory spleen colonies, but not of CFU‐s, as measured by different suicide techniques. During the log‐phase of cell growth in a lethally irradiated recipient, the injected CFU‐s and CFU‐tr proliferate at a higher rate. Active proliferation of CFU‐s and CFU‐tr has been demonstrated in long‐term bone‐marrow cultures by the hydroxyurea in‐vitro suicide assay. CFU‐tr may be the cause of artifactual effects during measurement of haemopoietic stem‐cell cycling by CFU‐s suicide methods.

Comparative effect of heme analogues on hematopoiesis in lymphoproliferative disorders.

Anemia is a common characteristic of lymphoproliferative disorders (LPD) and the impairment of blood formation in these disorders is not fully understood. Heme synthesis and the heme degradative enzyme heme oxygenase are critical to hematopoietic differentiation and disturbances may contribute to anemic states. Tin protoporphyrin (SnPP) is a potent inhibitor of heme oxygenase, and has proven to be a useful clinical agent. Bone marrow cells from seven patients with LPD were studied for their in vitro hemopoietic response to growth factors and SnPP. Heme oxygenase mRNA levels were determined by Northern blot analysis of bone marrow samples. Quantitation of hematopoiesis in cultures with erythropoietin or GM-CSF revealed adequate CFU-E, BFU-E and CFU-GM growth by LPD bone marrow. Inclusion of 10 μM SnPP in cultures was found to significantly enhance CFU-E/BFU-E growth by LPD marrows, whereas Zinc protoporphyrin had a marked inhibitory effect. Little or no effect by SnPP was seen on CFU-GM. In contrast, normal bone marrow cultures failed to show an enhanced response to 10 μM SnPP. Analysis of heme oxygenase mRNA levels revealed that LPD marrows had elevated expression of heme oxygenase mRNA as contrasted with normals. Furthermore, measurements revealed that heme oxygenase activity was markedly suppressed by SnPP in the LPD bone marrow cultures. Results lend further support to the importance of heme oxygenase in the differentiation process. Although LPD bone marrow cells may respond to erythropoietin in vitro, in stressed conditions where heme oxygenase is elevated, suppression of heme oxygenase may potentiate the erythropoietic response in this disease.

Self-Renewal Capacity and Clonal Succession of Haemopoietic Stem Cells In Long-Term Bone Marrow Culture

Abstract. The CFU‐s proliferative potential varied greatly during long‐term cultivation. Most of the CFU‐s in the cultures were represented by cells with low renewal capacity. Pre‐CFU‐s cells capable of producing multipotential colonies in methylcellulose, which contained CFU‐s with a high proliferative potential, were identified in the culture. In cultivation of a mixture of cells of different karyotype their ratio changed rapidly from week to week. the findings were consistent with the hypothesis that haemopoietic stem cells are maintained in the culture by the products of a small number of clones which arise and decline in succession, and that pre‐CFU‐s, but not the CFU‐s themselves, are clonogenic progenitors.

HAEMOPOIETIC STEM CELL (CFU) KINETICS IN THE CULTURE

Haemopoiesis continued for over 2 months in organ culture of embryonal mouse liver, and haemopoietic stem cells (CFUs) capable of DNA‐synthesis were found in it all that time. Between the 10th and 40th day the number of stem cells in the culture was sustained in a steady state. Both in normal and in regenerating adult bone marrow haemopoiesis ceased within a short time in the culture. Induction of proliferation in haemopoietic stem cells combined with undamaged or improved micro‐environment resulted in a little better maintenance of CFUs in the adult bone marrow culture, The results are discussed in the light of current concepts of haemopoietic stem cell regulation.

Induction of hematopoietic microenvironment by the extracellular matrix from long-term bone marrow cultures

SummaryExtracellular matrix (ECM) plays an important role in the regulation of hematopoiesis. The ECM obtained from murine long-term bone marrow cultures (LTBMCs) induces hematopoietic foci formation within 3 months after implantation under the murine renal capsule. The foci consist of approximately 3×106 hematopoietic cells and function for at least 11 months. The induced stroma contains transplantable precursors capable of transferring a hematopoietic microenvironment to secondary recipients, and is insensitive to the stroma-stimulating factor produced in recipient mice after irradiation. The ECM induces hematopoietic foci formation in chimeras irradiated by a dose which is lethal for most of the stromal precursors. These facts point to the differences observed between bone marrow stromal precursors and mesenchymal cells induced under the renal capsule. The foci contain bone, but its appearance is limited to early stages of foci growth, and depends on the dose of implanted ECM. Bone is not formed when the xenogeneic ECM from nonhematopoietic tissue is used as an inducer. In this case, the foci develop slowly and are observed only to the tenth month after implantation. The data obtained demonstrate a novel function of the ECM in the induction of a hematopoietic microenvironment.

Limited Proliferative Potential of Primitive Hematopoietic Stem Cells: Hematopoiesis by Clonal Succession

The hierarchy of hematopoietic stem cells (HSC) is represented by several categories of maturating pluripotent pro-genitors. Most of them are the members of transitional cell populations and, obviously, have no capacity for self-maintenance, i.e., are not capable of giving rise to self-replicating offspring with the same proliferative potential as the parent had [1]. The foundator of this hierarchy has not yet been identified. The most probable candidate at present is the cells supporting long-term hematopoiesis in vivo after repopulation of lethally irradiated or genetically defective W-mutant mice or in vitro in long-term culture. However, the self-renewal is also not proven for these cells, and hematopoiesis, at least in culture, occurs by clonal succession [2]. The experimental data support the hypothesis that even primitive HSC (PHSC) exhibit high, though limited, proliferative potential. This cell category is usually identified by competitive repopulation assay using a mixture of tested and standard cells identifiable by biochemical, immunological, karyological, or other markers [3]. Limiting dilution analysis based on the ability of small numbers of +/+ hematopoietic cells to cure anemia of W-mutant mice has been also used for the determination of PHSC [4–6].