Stromal cells from murine developing hemopoietic organs: comparison of colony-forming unit of fibroblasts and long-term cultures.

The adherent stromal layer in long-term marrow cultures is essential to the proliferation and differentiation of hemopoietic cells. Adhering cells are heterogeneous and morphologically not adequately characterized. Comparative morphological studies were conducted on adherent cells in short-term clonal assays and long-term cultures derived from liver and bone marrow. Liver and bone marrow at different developmental ages have different hemopoietic activities in vivo and in vitro, as tested via CFU-GM recovery in long-term cultures. Adherent cells from each organ were recovered at an age with high hemopoietic activity (fetal liver and adult bone marrow) and at an age with low hemopoietic activity (neonatal liver and bone marrow). The presence of macrophages, alkaline phosphatase, acid phosphatase, myeloperoxidase, sulfated and non-sulfated glycosaminoglycans (GAGs) and fibronectin was compared. For a given organ, CFU-f colonies showed characteristics similar to those of the confluent adherent stromal layer in long-term cultures. The presence of macrophages and GAGs (sulfated and non-sulfated) in the adherent layer were directly related to the hemopoietic activity. The amount of alkaline phosphatase-positive cells and the amount of fibronectin showed no correlation with the hemopoietic activity of the cultures.     

The dependence of the formation of stromal CFU-f colonies on the stimulating action of hematopoietic cells

CFU-f-derived stromal colony formation was accomplished in adherent marrow cell cultures (AMCC) with serum-rich medium. It turned out to require additional stimulation by hemopoietic feeder cells: by irradiated marrow cells and spleen cells if they possess megakaryocytes and platelets or by platelets from the blood. PDGF, EGF and IL-3 did not substitute the colony stimulating activity of feeder cells. Thymus, lymph node cells and blood leucocytes had no colony stimulating activity. At low oxygen concentrations which improve colony formation the stimulating activity of hemopoietic feeder cells was expressed, as well. Thus, CFU-f colony formation depends on stimulation by hemopoietic cells in addition to serum growth factors. In full populations of marrow cells the CFU-f colony formation is stimulated by marrow cells which accompany the CFU-f.     

Mechanisms of lymphocytic choriomeningitis virus-induced hemopoietic dysfunction.

Results of this study showed that lymphocytic choriomeningitis virus infection causes a marked activation of natural killer (NK) cells not only in the spleen but also in the bone marrow. This activity reached its peak at about day 3 of infection and declined after days 6 to 7. Enhanced NK cell activity was found to correlate with decreased receptivity for syngeneic stem cells in bone marrow and spleen, with the notable exception that decreased receptivity persisted longer in bone marrow. Treatment of infected recipients with anti-asialo GM1 (ganglio-N-tetraosylceramide) significantly increased the receptivity for syngeneic hemopoietic cells. These findings are consistent with the hypothesis that NK cell activation causes rejection of syngeneic stem cells, thus resulting in hemopoietic depression. To understand the mechanisms behind the prolonged decrease in bone marrow receptivity (and bone marrow function in the intact mouse) mentioned above, we followed the changes in the number of pluripotential stem cells (CFU-S) circulating in the peripheral blood and in endogenous spleen colonies in irradiated mice, the limbs of which were partially shielded. It was found that following a marked early decline, both parameters increased to normal or supranormal levels at about day 9 after infection. Because the bone marrow pool of CFU-S is only about 20% of normal at this time after infection, a marked tendency for CFU-S at this stage in the infection to migrate from the bone marrow to the spleen is suggested. It seems, therefore, that as NK cell activity declines, the spleen regains the ability to support growth of hemopoietic cells and the bone marrow resumes an elevated export of stem cells to the spleen. This diversion of hemopoiesis could explain both the long-standing deficiencies of the bone marrow compartment and the prolonged decrease in the receptivity of this organ.     

Analysis of sensitivity of stromal stem cells (CFU-f) from rat bone marrow and fetal liver to 5-fluorouracil

The sensitivity of stromal stem cells (CFU-f) from rat bone marrow and fetal liver to the cytotoxic effect of 5-fluorouracil (5-FU) was compared in vivo and in vitro. Cells from both tissues demonstrated a similar resistance to 5-FU in vitro; however, stromal stem cells from fetal liver proved notably more sensitive to 5-FU compared to marrow CFU-f in vivo. Cells forming colonies of different size were identified in stem cell populations from both tissues. Cells giving rise to small colonies had a higher resistance to 5-FU both in vivo and in vitro.     

Changes in the histostructure of functional activity of the immunocompetent organs in damaged portal blood supply of the liver

In CBA mice calibrated stenosis of the portal vein was produced. Liver and immunocompetent organs were morphologically analyzed. The total number of hemopoietic stem cells in the bone marrow was estimated by the colony-forming cells and in the spleen after immunization with sheep red cells by the plaque forming method. It is established that stenosis of the portal vein (on the average by 45% and 58%) produced the histostructural changes in the liver and in the immunocompetent organs. Expression of morphological changes depended on the time elapsed after operation and the degree of the portal vein stenosis. These changes were the most pronounced on the 16-17th day when stenosis of the portal vein was 58%. The character of the changes in the number of the hemopoietic stem cells in the bone marrow and in that of antibody-forming cells in the spleen depended on the degree of the liver damage. These changes increased with the degree of the liver histostructure damage. The maximal liver damage was accompanied by a decrease of these indices.     

An immunofluorescence analysis of the ontogeny of myeloid, T, and B lineage cells in mouse hemopoietic tissues

The population dynamics of granulopoietic cells, B-lineage cells, and T lymphocytes were analyzed by immunofluorescence in mouse hemopoietic tissues as a function of age. Mac-1+ myeloid cells were present on day 11 of gestation in the liver, where they peaked shortly after birth and declined subsequently. Waves of myeloid population growth began in spleen and bone marrow by days 15 and 19, respectively. Mac-1+ cells increased in number to relatively low plateau levels in spleen by the 3rd wk after birth, whereas in the bone marrow higher plateau levels were reached around 3 mo of age. The 14.8 monoclonal antibody was utilized as one marker of B-lineage precursor cells. 14.8+ cells were detected in the liver on day 11 of gestation, reached peak numbers during the first week after birth and decreased thereafter. On day 15 and 19, 14.8+ cells were found in spleen and bone marrow, respectively, and progressively increased in numbers to reach plateau levels in both sites by 3 mo of age. Mu+ pre-B cells appeared in significant numbers in the 13-day fetal liver, reached a peak shortly after birth, and disappeared from the liver by the end of the second postnatal week. Pre-B cells were found in the spleen and bone marrow on days 15 and 19, respectively. In the spleen pre-B cells reached peak values at birth and disappeared 2 wk later. In spite of the sequential appearance of mu+ pre-B cells in fetal liver, spleen, and bone marrow, their sIgM+ B cell progeny appeared in all these hemopoietic tissues on day 17 of gestation. In the liver, sIgM+ B cells reached their peak at birth and declined thereafter. In the spleen and bone marrow, B cells increased to plateau levels between 1 and 4 mo of age. Thy-1.2+ T cells were relatively late acquisitions in all three hemopoietic tissues. Finally, the expression of the 14.8 antigen by mu+ cells was examined as a function of gestational age. While pre-B cells from day-13 fetuses had no detectable 14.8 antigen, the antigen was weakly expressed on the vast majority of the mu+ pre-B cells by day 17 of gestation. Newborn liver cells expressing 14.8 antigen were found to include a small proportion of cells with peroxidase+ granules. Thus, demonstration of rearrangement and expression of immunoglobulin genes may be required for precise identification of cells of B lineage early in ontogeny.     

Clonogenic fibroblasts from the hematopoietic organs of the human embryo and fetus at different gestational ages

Fibroblast precursors of hemopoietic organs of 72 embryos and fetuses 5-27 weeks of age have been studied. The study has shown that the increase in the number of clonogenic fibroblasts took place in the bone marrow and spleen 2-3 weeks before the beginning of hemopoiesis, that is during the period of the highest hemopoietic stem cell concentration. These data suggest possible participation of stromal fibroblasts of hemopoietic organs in the formation of microenvironment for hemopoietic stem cell functioning.     

Proliferation and differentiation of hematopoietic stem cells in hypokinesia]

Using the method of exogenous cloning in vivo of the hemopoietic stem cells of the bone marrow and spleen in the femur and the spleen of mice it was shown that during hypokinesia the kinetics of the stem cells differed in both organs (the spleen and the bone marrow). Differentiation of transplanted stem cells from different sources was unchanged in the spleen, but stem cells of the bone marrow seeding in the femur changed the character of their differentiation in the direction of increase of the erythopoietic function, whereas stem cells of the spleen failed to alter the direction of differentiation.     

Cryopreservation of hemopoietic tissue aimed at reducing immunological reactivity

A method has been developed of decreasing immunologic activity of lymphocytes from spleen, lymph nodes, and bone marrow (freshly collected or frozen) with a high percentage of intact stem cells. In experiments in vivo on lethally irradiated mice, it was demonstrated that during combined transplantation to the recipients of preserved bone marrow from two genetically different donors, a rapid decrease or absence of the effect of inactivation of hemopoietic stem cells under the influence of allogeneic lymphocytes was observed in the mixed graft. When it is necessary to transplant large quantities of bone marrow from several genetically different donors, the use of cryopreserved bone marrow is preferable to freshly drawn marrow due to the higher proliferative activity and the decreased risk in the development of immunological reactions.     

Immunofluorescent study of the hemopoietic organs of xenogeneic mouse radiation chimeras]

An immunofluorescent study of hemopoietic organs in xenogenic (mouse-rat) radiation chimaeras has been carried out by means of specific antiserum against hemopoietic cells of the rat bone marrow. The presence of donor cells was tested at different times after the transplantation in the bone marrow, spleen, lymph nodes, thymus and liver of radiochimaeras. The transplanted cells were shown to populate all hemopoietic organs of the recipient, first of all tissues of the bone marrow type and, then, lymphoid organs. The donor (bone marrow) origin of the extramedullar foci of hemopoiesis in the liver was established.     

Developments in modern hematology.

In the past 40 years our concepts about hemopoiesis have been changed dramatically. The results of bone marrow transplantation into lethally irradiated mice since the mid-fifties suggested the existence of a hemopoietic stem cell, which was initially identified as a spleen colony forming cell (CFU-S). Later experiments showed that the stem cell compartment is rather heterogeneous and that the most primitive stem cell, unlike the CFU-S, has the ability for long-term engraftment of an irradiated recipient. Daughter cells of such primitive quiescent stem cells lose their capacity for self-generation gradually with each mitosis and become more and more committed to a specific differentiation lineage. In vitro culture techniques in a serum-free semi-solid medium enabled the establishment and analysis of specific hemopoietic growth factors. Such factors, which are essential for the maintenance, proliferation and differentiation of progenitor cells and the functional activity of mature cells can now be produced with recombinant DNA techniques in pure form and large quantities. Hemopoiesis requires an appropriate microenvironment, consisting of various stromal cell types and an extracellular matrix. Intercellular contacts, adhesion of cells and growth factors to the matrix molecules seem essential in the regulating action of this hemopoietic microenvironment. In long-term bone marrow cultures the development of a stromal hemopoietic microenvironment can facilitate long-term maintenance of stem cells and hemopoietic differentiation. For bone marrow transplantation and infusion of hemopoietic growth factors many clinical indications are well established and our possibilities to interfere in the regulation of hemopoiesis are still growing.     

Macromolecular insoluble cold globulin (MICG): a marker for pluripotential hemopoietic stem cells

In embryonic mice pluripotential hemopoietic stem cells (PHSC) originate in the yolk sac and migrate to the fetal liver and from there to the bone marrow. Hemopoietic cells from yolk sac and fetal liver also migrate to the thymic primordium, and within the thymic environment these prothymocytes differentiate into mature T cells. We have recently demonstrated that macromolecular insoluble cold globulin (MICG), a T cell marker, is synthesized and inserted into the plasma membrane of embryonic prothymocytes as soon as these cells appear in the early thymus. In addition, we have shown that MICG+ cells are present within the fetal liver before the thymus has fully formed. In the present study we show that pluripotential hemopoietic stem cells in the fetal liver and bone marrow have MICG on their surface and represent a subpopulation of these MICG+ cells. The implications of these findings in relationship to stem cell differentiation and isolation are discussed.     

The need of B-helpers for the T-lymphocyte regulation of the process of hematopoietic stem cell differentiation

We have detected and characterized a subpopulation of immunoregulatory cells, i.e., B-helpers capable to enhance the activity of Td-lymphocytes and controlling differentiation of syngeneic hemopoietic stem cells in mouse spleen and bone marrow. B-helpers found in the spleen and lymphatic nodes are resistant to radiation (at a dose of 6 Gr) but are impaired when irradiated at 9 Gr. Manifestation of the helper activity does not require either DNA or RNA synthesis but depends on protein synthesis and is mediated by soluble transmitter substances. Initial activation of B-helpers by lipopolysaccharide or alloantigens does not affect their helper functions. In the absence of T-lymphocytes B-cells do not affect differentiation of hemopoietic stem cells; interaction of B-helpers with differentiating Td-lymphocytes is not genetically restricted. Using preparative electrophoresis, we could isolate fractions of Td-lymphocytes which require or do not require B-helper cells in order to induce change in differentiation of hemopoietic stem cells from mainly erythroid to preferentially granulocyte pathway.     

Mock retroviral infection alters the developmental potential of murine bone marrow stem cells.

Retroviral vectors were used to introduce an activated ras gene into murine pluripotent hemopoietic stem cells. We attempted to reconstitute the hemopoietic system of lethally irradiated mice with isolated spleen colonies obtained in vivo after injection of infected bone marrow cells. Spleen colonies derived from infected bone marrow were inefficient in promoting long-term survival of irradiated hosts. This loss of reconstitutive capacity of spleen colonies was not due to the retroviral infection per se but to the in vitro culture of spleen colony precursors. Incubation for 24 h in the presence of fetal calf serum and interleukin-3 without virus-producing cells was sufficient to abolish completely the reconstitutive capacity of spleen colonies while maintaining both self-renewal and pluripotential capacities of spleen colony precursors. These results show that the in vitro manipulation of stem cells that is included in current protocols for retroviral infection can modify the developmental potential of these cells. This finding clearly indicates that the use of retroviral vectors can introduce a bias in the analysis of hemopoiesis.     

Pre-CFU-f: young-type stromal stem cells in murine bone marrow following administration of DNA inhibitors

Occurrence of young-type stromal stem cells (defined here as "pre-CFU-f") in murine bone marrow is reported in this study. Two consecutive intraperitoneal (i.p.) cytosine arabinoside (ara-C) injections were administered to C57B1 mice (2 X 200 mg/kg at 6-h intervals). Two days later the bone marrow was collected and assayed for colony-forming units-fibroblastoid (defined here as "CFU-f"). In additional experiments, ara-C-treated marrow was exposed in vitro to hydroxyurea (HU; "hydroxyurea killing test"), prior to plating, to establish the cycling state of stromal stem cells. In separate cultures of ara-C-treated marrow, replating of adherent cells was carried out up to quaternary sub-cultures. The results indicate ara-C-treated marrow produces approximately 20% "huge" fibroblastoid colonies (approximately 5 mm diameter versus 0.5-2 mm normal size); most stromal stem cells producing huge colonies are cycling cells; and adherent cells from primary ara-C-treated marrow cultures replated to secondary cultures produce adherent layers with double the number of cells than in the control secondary cultures. We conclude that the ara-C-treated murine bone marrow contains certain young-type cycling stromal stem cells which we refer to as pre-CFU-f. These stem cells produce huge fibroblastoid colonies in culture, indicating that they probably go through more cell cycles than CFU-f during the culture period. Alternatively, pre-CFU-f may have a higher self-replicative capacity than CFU-f.     

Host natural suppressor activity regulates hemopoietic engraftment kinetics in antibody-conditioned recipient mice

Resistance to semi-allogeneic or syngeneic hemopoietic stem cell engraftment can be reduced by treating the unirradiated host with anti-class I MHC antibody. In our previous studies we showed a direct correlation between such resistance and the level of natural suppressor (NS) activity in the host. Thus newborn mice that have high NS activity are very resistant to marrow engraftment, as are adults pretreated with CFA that increases NS activity in the bone marrow. We have now devised a method that allows us to follow hemopoietic engraftment kinetics within the marrow cavity itself by assaying individual CFU-granulocyte/macrophage progenitor cells for their host or donor origin over the immediate post-transplant period. By using this method, we find a close correlation between the rate of marrow engraftment and reduction in host NS activity. Marrow engraftment does not correlate with the reduction of either total host bone marrow cellular content or CFU-granulocyte/macrophage progenitor cell levels. NS activity is mediated by Thy-1-, partially radiosensitive, nylon wool nonadherent cells without NK activity. Adoptively transferred Thy-1-, irradiated spleen cells containing NS activity induced by pretreatment with CFA delayed engraftment kinetics in the marrow cavity. Thus hemopoietic engraftment in the marrow cavity appears to be controlled by an inhibitory regulatory activity that is reflected in the in vitro NS assay. These studies suggest new regulatory targets for selective host conditioning to eliminate resistance to marrow transplantation.     

Microenvironmental studies of pre-B and B cell development in human and mouse fetuses

Immunofluorescence techniques were used to trace the development of cells expressing mu heavy chains in humans and mice. IgM B cells were distinguished from pre-B cells by their additional expression of kappa or lambda light chains. Generation of pre-B and progeny B cells was evident in hemopoietic fetal liver and bone marrow, but not in thymus, heart, lung, spleen, kidney, and placental tissues. Pre-B and B cells, in a ratio of 2 to 1, were abundant in sections of hemopoietic liver and in bone marrow from 12- to 15-wk-old human fetuses, whereas these cells were rare in nonhemopoietic liver samples obtained beyond the 34th week. In mouse fetal liver mu+ cells appeared first around the 12th day of gestation and increased in frequency throughout the third trimester. On day 17 of gestation, kappa light chain expression by 1% of mu+ cells was noted, and the percentage of kappa+/mu+ cells increased progressively to more than 80% by 5 days after birth. Pre-B and B cells were interspersed among myeloid and more abundant erythropoietic cellular elements in the extrasinusoidal areas adjacent to hepatic cords. A loose clustering or "starburst" distribution pattern of pre-B cells became evident around day 17. These observations suggest a model for in situ generation of pre-B and progeny B cells in the hemopoietic fetal liver. In the midst of more numerous erythropoietic elements, immunoglobulin-negative precursors divide to generate a loose colony of mu+ pre-B cells that divide again before giving rise to a wave of IgM B cells.     

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

Different effect of testosterone on polypotential stem hematopoietic stem cells and immunocompetent B-lymphocytes]

A research was made to study the dynamics of the proliferative, colony-forming and migration capacity of stem hemopoietic cells in (CBA X C57Bl) F1 hybrid mice under the influence of testosterone propionate, 10 mg/100 g, as well as the migration of immunocompetent B lymphocytes from the bone marrow to the spleen and the accumlation of their progeny, antibody-producing cells, in the spleen. The immunodepressive effect of testosterone was manifested by a decrease in the migration of B cells and the number of antibody-producing cells in the spleen. On the contrary, testosterone had a stimulating effect on the functional activity of stem hemopoietic cells, increasing their proliferation and migration. Under conditions of the suppressed erythropoietic differentiation of multipotent stem hemopoietic cells the injection of testosterone resulted in an increase in the number of antibody-producing cells in the spleen. This suggests that the stimulation of erythropoiesis and immunosuppression, induced by testosterone, are interconnected and determined by the direct action of the hormone on the cellular cycle of the stem cells, as well as by their prevailing differentiation towards the erythroid series, resulting in the decrease of their differentiation into B cells.     

Ontogeny of terminal deoxynucleotidyl transferase-positive cells in lymphohemopoietic tissues of rat and mouse.

The ontogeny of hemopoietic cells which contain the enzyme terminal deoxynucleotidyl transferase (TdT) was studied in rats and mice. During fetal life, TdT-positive cells were first detected in the thymus, where they appeared on or about day 17 of gestation. TdT-positive cells were not found in fetal liver, spleen, or bone marrow, but appeared in bone marrow and spleen on the day after birth. In the rat, peak levels of TdT-positive cells were attained at 3 to 4 weeks of age in thymus, bone marrow, and spleen, accounting for 67, 3.9, and 2.3% of nucleated cells, respectively. The percentages of TdT-positive cells in thymus and bone marrow decreased gradually thereafter, whereas, TdT-positive cells in spleen were no longer detectable by 7 weeks of age. Normal percentages of TdT-positive cells were found in bone marrow and spleen from neonatally thymectomized rats and congenitally athymic (nu/nu) mice. Dexamethasone treatment resulted in a marked decrease in TdT-positive cells. The results are discussed with respect to the putative role of TdT-positive hemopoietic cells as thymocyte progenitors.