COMPARTMENTS AND CELL FLOWS WITHIN THE MOUSE HAEMOPOIETIC SYSTEM

Two chromosomally distinguishable haemopoietic cell populations were injected into lethally irradiated syngeneic recipients. The presence or absence of the T(14;15)6Ca reciprocal translocation (indicated by T6 marker chromosomes) did not affect the proliferation of a population. Wide disparities were found in the proportions of the two donor cell populations between animals and between the right and left femora of individual animals. This suggests (i) that there is, at most, a very limited interchange of proliferating cells and their precursors between the marrow of different bones; and (ii) that the number of clones proliferating in the bone marrow at any one time must be rather small; there was evidence that this number depended in part on the number of haemopoietic cells injected. Exchange between the mitotically active cell populations of spleen, thymus, lymph nodes and bone marrow was also limited, as shown by significant disparities in the proportions of the two donor populations proliferating in the different tissues of individual mice.     

Mechanisms of haemopoietic stem cell proliferation control

The control of stem cell (CFU-S) proliferation is mediated by short-range acting factors which can be detected by the proliferation modifying activities present in media conditioned by haemopoietic cells. A specific inhibitor of stem cell proliferation is obtained from haemopoietic tissue containing minimally proliferating CFU-S, whilst stimulatory material is obtained from cell suspensions containing rapidly proliferating CFU-S. Used competitively, these factors, which are detected in different molecular weight range fractions, manipulate the rate of CFU-S proliferation in a manner compatible with a physiological control mechanism. In addition, a long-term bone marrow culture system has been shown to provide an in vitro model of stem cell control. Fractionation of cell populations from haemopoietic tissues reveals marked concentration differences of the CFU-S proliferation modifying activities depending on the proliferative state of the CFU-S. However, irrespective of whether the tissue contains stem cells that are actively or minimally proliferating, both stimulatory and inhibitory activities are detected. From dose-response studies it is concluded that stem cell proliferation is controlled by an appropriate balance of stimulatory and inhibitory factors which, however, are not produced by the stem cells themselves.     

COMPARTMENTS AND CELL FLOWS WITHIN THE MOUSE HAEMOPOIETIC SYSTEM II. ESTIMATED RATES OF INTERCHANGE

Part-body irradiated CBA mice were injected with CBA-T6 bone marrow. In this way a predominantly donor population was established in the femora while the marrow of the humeri remained largely (average 94 %) of host origin. In animals examined cytologically up to 2 years later, no tendency was observed for the proportion of donor cells in the humeri to increase. Splenectomy had no effect on this. When femoral bone marrow from the experimental mice was injected into lethally (whole-body) irradiated recipients, cells originating from the primary host repopulated the lymph nodes to a disproportionate extent. Equilibration between the cell populations of femora and humeri occurred after re-exposure to 600 rad whole-body irradiation, but not after 100 rad or 350 rad; thus, regeneration of damaged bone marrow involved a significant contribution from extrinsic stem cells only after the highest dose of radiation. The data are compatible with an inflow of at most ten effective stem cells per humerus per day from the blood, and suggest a much lower figure. This means that few if any of the stem cells of peripheral blood enter the bone marrow and found haemopoietic clones. Evidence is adduced for the existence of a proliferating lymphoid sub-population in the bone marrow, contributing some 5–10% of the observed mitoses. The mitotic cells in the lymph nodes are replaced from marrow-derived progenitors at an estimated rate of 4–5 %/day. The relevant data for the thymus are more variable, but suggest an average figure of 8–11 %/day. Earlier data from mouse parabionts suggest a lower rate of inflow to the thymus.     

In vivo studies on the regeneration kinetics of enriched populations of haemopoietic spleen colony-forming cells from normal bone marrow

Functional properties of mouse haemopoietic spleen colony-forming cells, enriched 40- to 80-fold, from normal bone marrow were studied. It was found that: (1) the number of partially purified CFU-s (colony forming unit-spleen) required to rescue lethally irradiated mice was similar to the number of normal unfractionated bone marrow CFU-s giving the same level of protection; (2) the homing of partially purified CFU-s was similar to that of CFU-s from unfractionated bone marrow; (3) the regeneration of CFU-s in spleen was similar for enriched and unfractionated cell populations between 4 and 11 days after transplantation. In contrast, the rate of regeneration of CFU-s in femur was slower with enriched progenitor cells than with unfractionated bone marrow. The growth rate in femur, however, could be restored to normal by injecting freshly isolated syngeneic thymocytes with the enriched CFU-s population. The results indicate that the partially purified CFU-s are by themselves functionally normal and show that the rate of CFU-s repopulation in bone marrow can be affected by cell types other than spleen colony-forming cells.     

The effect of dextran sulphate on CFU-S and nucleic acids in blood and haemopoietic tissues in irradiated mice.

The effect of synthetic polyanion dextran sulphate on the development and recovery of radiation-induced haemopoietic damage in mice was investigated. Dextran sulphate (mol. wt. 500,000 D) in the dose of 40 mg.kg-1 of body weight was injected i.p. 3 days before single total body irradiation with a dose of 7.8 Gy gamma-rays. The animals were examined from hour 6 to day 26 after irradiation, i.e. from hour 78 to day 29 after DS-treatment. In irradiated mice DS-pretreatment showed some positive effect on the CFU-S number in bone marrow (less in spleen and blood), bone marrow cellularity, attenuated the radiation-induced changes of erythrocytes (number, MCV) and of RNA concentration in blood. The changes of other parameters (spleen cellularity, liver CFU-S, leukocyte count and DNA concentration in blood) were the same as in unprotected animals. In conclusion, we can say that DS-pretreatment had a beneficial effect on the recovery of radiation-induced damage of erythropoiesis but not on granulopoiesis or lymphopoiesis.     

A COMPARATIVE STUDY OF THE REPOPULATING POTENTIAL OF GRAFTS FROM VARIOUS HAEMOPOIETIC SOURCES: CFU REPOPULATION

The growth rate of the CFU populations in spleens and femora has been studied in irradiated mice injected with cell suspensions, containing equivalent number of CFU, from various sources. The doubling times are shown to be dependent upon the source of the cells. Grafts of bone marrow, spleen and foetal liver cells produced doubling times in the spleen of approximately 25, 19 and 16 hr respectively. Grafts of marrow-derived and spleen-derived spleen colony cells both gave rise to CFU doubling times lower than those of the corresponding primary grafts (approx. 33 and 26 hr respectively in the spleen). In the case of both bone marrow and spleen grafts the CFU population growth was shown to be independent of the size of the graft. A hypothesis is advanced which invokes at least a dual population of CFU, having different doubling times, different seeding capacities in the haemopoietic tissues following i.v. injection and present in different proportions in the various haemopoietic tissues.     

Differentiation and grafting of haemopoietic stem cells from early postimplantation mouse embryos

Haemopoietic stem cells evidently arise in early post-implantation mouse embryos at day 6 of gestation, a day earlier than previously thought (Moore & Metcalf, 1970). Disaggregated embryonic cells were injected into mice given a lethal dose of X-irradiation. The presence of donor haemoglobin (Whitney, 1978) and donor lymphocytic glucose phosphate isomerase (GPI) (Siciliano & Shaw, 1976) to detect donor erythrocytes and lymphocytes, respectively, were monitored by starch gel electrophoresis. The presence of donor cells was also assessed by using donor embryos carrying the T6 marker chromosomes. Decidual cells dissected free of embryos did not colonize any recipients. Disaggregated cells from early mouse embryos first colonized the liver and then repopulated the haemopoietic systems of recipients, producing adult donor haemoglobin within 2-3 days and donor GPI within 3-5 days. 80% of grafted X-irradiated recipients survived and donor markers were found in each of them. All nongrafted controls died within 14 days of X-irradiation and none of them showed donor markers. Disaggregated embryonic cells could be grafted across major histocompatibility barriers unlike adult bone marrow. Haemopoietic stem cells could not be identified in disaggregated cells from embryos aged less than 6 days gestation.     

Effects of human leukocyte conditioned medium on mouse haemopoietic progenitor cells.

Medium conditioned by human peripheral blood leukocytes (HLCM) was studied for its in vitro effects on haemopoietic progenitor cells (CFU-s and CFU-c) present in mouse bone marrow. HLCM has poor colony stimulating activity in semi-solid cultures of mouse bone marrow cells, but invariably increases the number of colonies obtained in the presence of plateau levels of semi-purified colony stimulating factor (CSF). In liquid cultures, HLCM appears to contain a potent initiator of DNA synthesis in CFU-s, an activity which coincides with an increased CFU-s maintenance and causes a three- to four-fold increase in CFU-c number. It is apparent from this study that HLCM, in addition to stimulating colony formation in cultures of human bone marrow cells, has a profound in vitro effect on primitive haemopoietic progenitor cells of the mouse, which cannot be attributed to CSF.     

The enhancement of haemopoietic stem cell recovery in irradiated mice by prior treatment with cyclophosphamide.

Studies are reported of the enhancement of stem cell recovery following whole body irradiation as a result of prior administration of cyclophosphamide. It is shown that the much larger enhancement of regeneration observed for the hosts own surviving stem cells, compared to the regeneration of injected bone marrow stem cells, is due to the different numbers of stem cells initiating the regeneration in conjunction with the time course of stem cell regeneration. The results show that the environmental changes produced by cyclophosphmide greatly enhance haemopoietic recovery even though at the dose used this agent is relatively toxic to stem cells. Furthermore it has been shown that the level of stem cell regeneration is nearly independent of the gamma-ray dose in the range 3-8 gray (300-800 rad). If human bone marrow should respond similarly it follows that regeneration produced by cytotoxic drugs administered prior to radiation embodies a considerable safety factor as far as recovery of the haemopoietic system is concerned.     

Effect of haemopoietic stem-cell proliferation regulators on early and late spleen colony-forming cells

An inhibitor and stimulator of CFU-s proliferation can be obtained from haemopoietic tissue containing, respectively, relatively quiescent CFU-s (e.g. normal bone marrow) and proliferating CFU-s (e.g. regenerating bone marrow). Their effects on the proliferative behaviour of steady-state and regenerating marrow CFU-s, which produce colonies 7, 10 and 12 days post-transplantation have been investigated. The results demonstrate changing sensitivities of CFU-s to inhibitor and stimulator as they progress through a developmental age structure, 'Older' CFU-s (producing early spleen colonies) are more sensitive to stimulator, 'Younger' CFU-s (producing late spleen colonies) are more sensitive to inhibitor.     

Role of cytokines and extracellular matrix in the regulation of haemopoietic stem cells

The understanding of molecular mechanisms regulating the formation, growth and differentiation of haemopoietic stem cells has advanced considerably recently. Particular progress has been made in defining the cytokines, chemokines and extracellular matrix components which retain and maintain primitive haemopoietic cell populations in bone marrow. Furthermore, signal transduction pathways that are critical for haemopoiesis, both in vivo and in vitro, and that are activated by cytokines have also been identified and further characterised. The importance of these processes has, this year, been exemplified by the phenotypes of mice deficient in key signal transduction proteins and the discovery that mutations in the component proteins of some signalling pathways are linked to human diseases. Significant advances in understanding the molecular mechanisms for mobilisation of stem cells from bone marrow have also been made this year; this has potential importance for bone marrow transplantation.     

Proliferation inhibition of murine pluripotent haemopoietic stem cells by interferon or poly I:C

The effect of mouse serum interferon (IF) in vitro and an inducer in vivo on the proliferation of a pluripotent stem cell population with high turnover rate was studied. Proliferation rate was characterized by the number of CFUs in the S phase of the cell cycle. Increased proliferation of bone marrow stem cell populations was produced either by irradiating the donor mice with 3.36 Gy (336 rad) 60Co-gamma rays 7 days before the experiment or by incubating normal bone marrow cells with 10(-11) M concentration of isoproterenol. IF considerably reduced the number of CFUs in S phase in both cases without reducing the CFUs content of the samples. Injection of IF inducer (4 mg/kg poly I:C) into regenerating mice also inhibited the proliferation of CFUs without decreasing the femoral CFUs level. Regeneration kinetics of CFUs from irradiated poly I:C-treated mice ran parallel with that of irradiated untreated animals but showed a characteristic delay corresponding to approximately one CFUs doubling. A transient, non-cytotoxic proliferation inhibitory effect of IF or IF inducer is, therefore, proposed.     

PROLIFERATION OF ERYTHROID AND GRANULOCYTE PROGENITORS IN THE SPLEEN AS A FUNCTION OF STEM CELL DOSE

A study of the kinetics of cellular proliferation, in the morphologically unrecognizable haemopoietic progenitor cell compartment, as a function of injected CFU-S dose has been carried out in the spleens of lethally X-irradiated mice using ³H-TdR labelling. Amplification in this proliferating cell compartment was observed to decline as CFU-S dose increased. The number of divisions in the differentiated line arising from CFU-S up to the first appearance of recognizable erythroid precursors were calculated to be 9.2, 12.5, 15 and 17 for the 2, 0.35, 0.05 and 0.007 femur equivalent doses respectively. The growth of cell populations arising from CFU-S was biphasic, with a rapid initial phase having a doubling time of about 6.3 hr, and a slow phase of doubling time around 1 day. Analysis of the rapid phase by the FLM method gave a cycle time of 5.6 hr. Recognizable labelled erythroid precursors were detected at the same time as, or just after, the change in slope of the growth curve. Significant numbers of proliferating (labelled) granulocytes only appeared in the spleens of animals receiving the higher marrow doses (2 and 0.35 femur). The erythroid to granulocyte ratio was also a decreasing function of marrow dose.     

Allogeneic chimerism in scid mice after neonatal transfer of bone marrow.

Allogeneic chimeras are valuable tools for studies of complex immune cell interactions in vivo. Mice with severe combined immune deficiency (scid) should be ideal hosts for chimerism with allogeneic bone marrow cells as these animals lack mature T and B lymphocytes capable of reacting against donor alloantigens. However, it has been difficult to achieve full reconstitution of adult scid mice even using coisogenic bone marrow grafts without prior irradiation of the recipient. We explored ways to generate complete reconstitution of scid mice with allogeneic bone marrow. Unirradiated adult scid recipients of allogeneic bone marrow were only marginally reconstituted. Adult scid mice pretreated with 250 R were reconstituted with allogeneic bone marrow as measured by serum IgM concentration, peripheral lymphoid cellularity, and mitogen responses, but a potentially important immunologic deficit was found in these mice: 250 R caused a 70% loss of scid macrophages and dendritic cells which persisted at least 5 months. By contrast, when scid mice were injected i.p. with allogeneic bone marrow within the first 24 h after birth, rapid and complete reconstitution of both T and B cell lineages was achieved, and the animals had APC that were both donor and host in origin. Considering the extent and duration of engraftment (43 wk) by allogeneic cells in neonatally transplanted scid mice, it was anticipated that their bone marrow would be chimeric. However, the bone marrow contained very few donor-derived cells, suggesting that lymphopoiesis may be taking place in other organs in these chimeras.     

Age dependence of the number of the stem cells in haemopoietic tissues of rats.

The number and concentration of haemopoietic stem cells in the femoral bone marrow and spleen of Wistar rats of different ages were investigated. Stem cells were assayed by the spleen colony technique in irradiated rat recipients. The ability of the recipient spleen to harvest transplanted tissue as a macroscopic colony was found to be dependent on the recipient's age. Changes with senescence were observed also in the concentration and the size of the stem cell compartment both in the marrow and spleen. No differences were demonstrated in the seeding of transplanted colony-forming units into the spleen of recipients of 1 and 4 months of age. A rats-mice strain difference in the effect of senescence on the haemopoietic stem cells is discussed.     

Modification of exogenous haemopoietic spleen colony formation in irradiated mice by antisera against mouse thymocytes and mouse brain.

Treatment of donor bone marrow in vitro with both anti-thymocyte serum (ATS) and anti-mouse brain serum (RAMBS) inhibits the formation of haemopoietic spleen colonies in irradiated and reconstituted mice. This activity of the antisera may be completely (ATS) or partly (RAMBS) eliminated by absorption with thymocytes. The effect of the antisera is complement-independent. Most likely it depends on the existence and functionality of phagocytes (macrophages) in the recipients.     

Effect of selective T cell depletion of host and/or donor bone marrow on lymphopoietic repopulation, tolerance, and graft-vs-host disease in mixed allogeneic chimeras (B10 + B10.D2----B10)

Reconstitution of lethally irradiated mice with a mixture of T cell-depleted syngeneic plus T cell-depleted allogeneic bone marrow (B10 + B10.D2----B10) leads to the induction of mixed lymphopoietic chimerism, excellent survivals, specific in vivo transplantation tolerance to subsequent donor strain skin grafts, and specific in vitro unresponsiveness to allogeneic donor lymphoid elements as assessed by mixed lymphocyte reaction (MLR) proliferative and cell-mediated lympholysis (CML) cytotoxicity assays. When B10 recipient mice received mixed marrow inocula in which the syngeneic component had not been T cell depleted, whether or not the allogeneic donor marrow was treated, they repopulated exclusively with host-type cells, promptly rejected donor-type skin allografts, and were reactive in vitro to the allogeneic donor by CML and MLR assays. In contrast, T cell depletion of the syngeneic component of the mixed marrow inocula resulted in specific acceptance of allogeneic donor strain skin grafts, whether or not the allogeneic bone marrow was T cell depleted. Such animals were specifically unreactive to allogeneic donor lymphoid elements in vitro by CML and MLR, but were reactive to third party. When both the syngeneic and allogeneic marrow were T cell depleted, variable percentages of host- and donor-type lymphoid elements were detected in the mixed reconstituted host. When only the syngeneic bone marrow was T cell depleted, animals repopulated exclusively with donor-type cells. Although these animals had detectable in vitro anti-host (B10) reactivity by CML and MLR and reconstituted as fully allogeneic chimeras, they exhibited excellent survival and had no in vivo evidence for graft-vs-host disease. In addition, experiments in which untreated donor spleen cells were added to the inocula in this last group suggest that the presence of T cell-depleted syngeneic bone marrow cells diminishes graft-vs-host disease and the mortality from it. This system may be helpful as a model for the study of alloresistance and for the identification of syngeneic cell phenotypes, which when present prevent engraftment of allogeneic marrow.     

Proliferation of erythroid and granulocyte progenitors in the spleen as a function of stem cell dose.

A study of the kinetics of cellular proliferation, in the morphologically unrecongizable haemopoietic progenitor cell compartment, as a function of injected CFU-S dose has been carried out in the spleens of lethally X-irradiated mice using 3H-TdR labelling. Amplification in this proliferating cell compartment was observed to decline as CFU-S dose increased. The number of divisions in the differentiated line arising from CFU-S up to the first appearance of recognizable erythroid precursors were calculated to be 9-2, 12-5, 15 and 17 for the 2, 0-35, 0-05 and 0-007 femur equivalent doses respectively. The growth of cell populations arising from CFU-S was biphasic, with a rapid initial phase having a doubling time of about 6-3 hr, and a slow phase of doubling time around 1 day. Analysis of the rapid phase by the FLM method gave a cycle time of 5-6 hr, Recognizable labelled erythroid precursors were detected at the same time as, or just after, the change in slope of the growth curve. Significant numbers of proliferating (labelled) granulocytes only appeared in the spleens of animals receiving the higher marrow doses (2 and 0-35 femur). The erythroid to granulocyte ratio was also a decreasing function of marrow dose.     

Effect of bone morphogenetic protein-6 on haemopoietic stem cells and cytokine production in normal human bone marrow stroma

Normal human bone marrow stroma cells include stem cells for both haemopoietic and osteochondrogenic lineages and express both bone morphogenetic protein (BMP) type I and type II receptors. As a member of the TGF-beta super-family, BMP-6 binds to both BMP type I and type II receptors and is involved in the developmental processes of renal and hepatic systems as well as of human foetal intestine. Also, BMP-6 induces osteoblastic differentiation of pluripotent mesenchymal cells and is an autocrine stimulator of chondrocyte differentiation. The present study was carried out to investigate the effect of BMP-6 on human cobblestone-area-forming cells (CAFC), that represent the functional primitive repopulating haemopoietic stem cell in long-term bone marrow culture. Also, the effect of BMP-6 on marrow stroma production of interleukin-6, -11 and their common receptor gp130 that is expressed in haemopoietic stem cells and is indispensable for their proliferation and tri-lineage differentiation was examined. Moreover, the effect of BMP-6 on marrow stroma release of soluble adhesion molecule VCAM-1 mediating the primitive haemopoietic stem cell adhesion to marrow stroma was examined. The number of CAFC was significantly reduced after BMP-6 treatment from 88+/-10 per 10(5)cells in control cultures in a dose dependent manner to only 48+/-3 per 10(5)cells in 50 ng/ml BMP-6-treated cultures, P< 0.01. Quantitative ELISA measurement revealed 50 ng/ml BMP-6 was able to significantly reduce IL-6 and IL-11 production from marrow stroma, P< 0.01. Also, BMP-6 significantly increased soluble gp130 release by 7.4-fold in 50 ng/ml BMP-6-treated marrow stroma cultures. The profound rapid increase in this natural antagonist of human IL-6 cytokine family may reduce the gp130 signaling. Also, the soluble VCAM-1 released increased by two-fold in 50 ng/ml BMP-6-treated marrow stroma cultures. The marked increase in the soluble form may exert an antagonist effect on the function of VCAM-1 (ligand for VLA4). Recently, blocking the VLA4/VCAM-1 adhesion pathway was shown to mobilise haemopoietic CD34 positive cells in normal individuals. Also, we previously observed a significantly lower expression of VLA4 (CD49d) on G-CSF-mobilised blood CD34 positive cells than on bone marrow CD34 positive cells before mobilisation in the same normal donors. Since BMP are currently being used in clinical trials for bone repair and fracture healing, the present results suggest a possible role for BMP-6 in mobilising CD34 positive cells for transplantation. Further in vitro tests are required to evaluate this potential mobilising role of BMP-6 in human long-term bone marrow culture.