The ability of induced osteo-progenitor cells to maintain and rebuild long-term ectopic osteo-hematopoietic foci in vitro.

Tooth matrix particles were implanted s.c. into mice. In six to nine months, fully developed, induced osteo-hematopoietic foci complete with a cortex and central marrow cavity were formed. The cells from the induced foci were serially retransplanted under the renal capsule of syngeneic recipients. The focus formation was observed during three consecutive passages. The effect was not due to secondary induction. Our data show that the induced osteogenic cells are capable of long-term maintenance, remodeling and recurrent rebuilding of hematopoietic stroma without an additional application of the inductor.

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.

Origin of hemopoietic stromal progenitor cells in chimeras.

Intravenously injected bone marrow cells do not participate in the regeneration of hemopoietic stromal progenitors in irradiated mice, nor in the curetted parts of the recipient's marrow. The hemopoietic stromal progenitors in allogeneic chimeras are of recipient origin. The adherent cell layer (ACL) of long-term cultures of allogeneic chimera bone marrow contains only recipient hemopoietic stromal progenitors. However, in ectopic hemopoietic foci produced by marrow implantation under the renal capsule and repopulated by the recipient hemopoietic cells after irradiation and reconstitution by syngeneic hemopoietic cells, the stromal progenitors were of implant donor origin, as were stromal progenitors of the ACL in long-term cultures of hemopoietic cells from ectopic foci. Our results confirm that the stromal and hemopoietic progenitors differ in origin and that hemopoietic stromal progenitors are not transplantable by the intravenous route in mice.

Self-renewal capacity and clonal succession of haemopoietic stem cells in long-term bone marrow culture.

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.

Hemopoietic stromal precursors in long-term culture of bone marrow: I. Precursor characteristics, kinetics in culture, and dependence on quality of donor hemopoietic cells in chimeras.

The adherent cell layer of long-term Dexter type bone marrow culture contains hemopoietic stromal precursors capable of transferring the hemopoietic microenvironment on transplantation in vivo. The foci of ectopic hemopoiesis that are created correspond to normal bone marrow in the character of the maintained hemopoiesis. Radiosensitivity of the cultured stromal precursors does not differ from preculture sensitivity. Stromal precursors lose osteogenic potential after 5-6 wk in culture, but are capable of creating stable foci of ectopic hemopoiesis composed of up to 10(8) nucleated hemopoietic cells. These results prove that bone is not an essential component of the hemopoietic environment in ectopic foci. In chimeras exposed to equal doses of radiation but reconstituted by freshly isolated or repeatedly passaged bone marrow, the content of stromal precursors is lower when hemopoietic cells of diminished self-maintenance are used. These data suggest the existence of a hemopoietic stromal precursor hierarchy with at least two levels of precursor differentiation.

Hemopoietic stromal precursors in long-term culture of bone marrow: II. Significance of initial packing for creating a hemopoietic microenvironment and maintaining stromal precursors in the culture.

Conversion of bone marrow cells to a single cell suspension prevents them from creating a hemopoiesis maintaining adherent cell layer (ACL) in culture. The ACL of such cultures is devoid of hemopoietic stromal precursors capable of transferring the hemopoietic microenvironment on implantation under the renal capsule of syngeneic mice. The regeneration of ACL after injury is possible in 1-wk-old, but not older, Dexter-type bone marrow cultures. The results prove the significance of the initial packing of the bone marrow cells in the differentiation of the hemopoietic stromal precursors. These data contradict previous reports which concluded that the structural orderliness of stromal tissue need not be preserved for the hemopoietic microenvironment to be transferred.

Self-maintenance ability of circulating hemopoietic stem cells.

In adult mice, circulating hemopoietic stem cells (CFUs) and their progeny that repopulate an ectopic hematopoietic site possess a self-maintenance potential that is less than that of resident stem cells. This is manifested by a decrease in the content of daughter CFUs in 11-day spleen colonies produced by circulating CFUs, compared to those produced by resident CFUs. Study of the CFUs content in 139 individual spleen colonies showed that circulating CFUs produce the entire spectrum of colonies, both those with a low and those with a high CFUs content. However, the proportion of colonies that contained 10 or fewer CFUs was much higher among the colonies produced by circulating CFUs (60-70%) than by the resident CFUs (less than 10%). The lower self-maintenance of circulating CFUs, compared to that of resident CFUs, is also confirmed by direct comparison of the overall regeneration of hemopoietic populations derived from blood and bone marrow by means of chromosome markers. The embryonal circulating and resident CFUs (from the liver) do not differ in self-maintenance. In adult mice, intravenously infused circulating CFUs leave the circulation no more slowly than do the resident CFUs.

Self-maintenance ability and kinetics of haemopoietic stroma precursors.

The use of repeated femoral curettages and repeated passages of ectopic haemopoietic foci had demonstrated the capacity of stromal precursor cells for repeated formation of haemopoietic microenvironment. During this process the stromal precursors undergo no less than ten to twelve mitoses. Precursors of bone marrow stroma proliferating slowly, if at all, in normal mice are triggered into cell cycle, as raevealed by suicide methods, during formation of the ectopic haemopoietic focus.

Role of bone marrow stroma in hemopoietic stem cell regulation.

Bone marrow plugs from C57Bl mice were irradiated with 1500 rad, implanted under the kidney capsule of (CBA x C57Bl)F1 hybrid mice and reimplanted one month later in identical F1 hybrid mice. Foci of ectopic hemopoiesis formed in which all hemopoietic cells were of recipient origin, while the stroma was from the donor bone marrow. The hybrid resistance in such foci was of donor origin. Implantation of 8--10 femoral plugs under the capsule of each kidney produced large foci of ectopic hemopoiesis containing thousands of hemopoietic stem cells (CFUS). The sum total CFUS content in such mice was two- to four-fold than in normal mice in steady-state hemopoiesis. It is concluded that the regulation of stem cell proliferation and total content in the body is governed by the stroma of hemopoietic organs.

Hybrid resistance to parental bone marrow transplantation: adaptive modification of hemopoietic stem cell in a nonsyngeneic environment.

Defective growth of parental bone marrow in an F1 hybrid is associated with delay of the exponential growth phase of injected hemopoietic stem cells rather than with their rejection. This is demonstrated both by parental hemopoietic stem cell kinetics in the irradiated hybrid and by the increase in the number of spleen colonies with time after hemopoietic cell injection. After passage through an F1 hybrid the parental hemopoietic stem cells acquire ability for better growth in the same F1 host. This phenomenon, which we designated "adaptive modification of hemopoietic stem cells," is associated with the appearance, on their surface, of histocompatibility molecules carrying H-2 determinants of the recipient. Treatment of the modified cells with antiserum against the second parental strain abrogates the state of adaptive modification.

Immunologic study of T and B lymphocytes in thymectomized monkeys after irradiation and autologous bone marrow transplantation.

Rhesus monkeys were exposed to total-body gamma-irradiation (60Co) with 700R following adult thymectomy, and then protected with autologous bone marrow. Transplantation immunity in TxIBM animals was impaired: skin allografts survived for 19 to 38 days. The percentage of T and B lymphocytes studied by the rosette technique and immunofluorescent staining for Ig did not substantially change when examined in early as well as late periods after TxIBM. Functional capacities of lymphocytes were measured in vitro by stimulation with phytohaemagglutinin and with allogeneic cells in mixed lymphocyte cultures. MLC reactivity in TxIBM monkeys did not recover in the early and later periods following treatment. PHA responsiveness was retained in both these periods. These findings together with the results obtained in the analysis of unstable chromosomal aberrations suggested that PHA-responsive and MLC-reactive cell subpopulations of monkey blood lymphocytes differed in the degree of thymus dependence.

Transplantation of allogeneic bone marrow from thymus-deprived monkeys and certain characteristics of "graft-versus-host" cells.

Transplantation of allogeneic bone marrow both from normal and thymus-deprived donors (thymectomized lethally irradiated and restored with autologous bone marrow) was followed 6--7 days later by the appearance of large numbers of GVH cells in bone marrow and peripheral blood which were one of the characteristic features of acute GVHR. GVH cells isolated from peripheral blood 7 days after transplantation showed a high labelling index (about 30%) after 40 min incubation with 3H-thymidine. The prolongation of incubation failed to increase the proportion of labelled cells due to the low viability of GVH cells. The results obtained with T and B cell tests suggested that GVH cells had the properties of T lymphocytes: about 60% of the cells could form rosettes with non-sensitized sheep red blood cells; there were virtually neither Ig-bearing cells nor cells with receptors for C3 and Fc portion of Ig.