Synchrony of bone marrow proliferation and maturation as the origin of cyclic haemopoiesis.

Cyclic haemopoiesis in Grey Collie dogs is characterized by stable oscillations in all haemopoietic lineages. It is proposed that in these animals, in contrast to normal animals, the maturation process of haemopoietic (in particular granuloid) cells from the primitive progenitors to the functional cells is characterized by an abnormally strong synchrony. It is conjectured that the marrow maturation time has a very small variance compared with non-cyclic normal dogs. With a mathematical model of haemopoiesis it is shown that small fluctuations are amplified via regular feedback processes such that stable granuloid oscillations are established. Erythroid oscillations are induced indirectly by granuloid feedback to the stem cell pool. The model calculations further show that the synchrony hypothesis of bone marrow maturation can quantitatively explain the following experimental results: (1) the maintenance of stable cycles of granuloid and erythroid bone marrow and blood cells with a period of approximately 14 d; (2) the disappearance of granuloid and erythroid cycles during the administration of the colony stimulating factor rhG-CSF; (3) the reappearance of oscillations when the administration of CSF is discontinued; (4) the cessation of cycles during endotoxin application; and (5) the persistence of cycles during erythroid manipulations (bleeding anaemia, hypoxia, hypertransfusion). We therefore conclude that cyclic haemopoiesis is not caused by a defect in the regulatory control system but by an unusual maturation process.     

Characteristics of the dynamics of hematopoietic precursor cells cloned in diffusion chambers and recorded in experiments on mice and dogs irradiated in median lethal doses]

In experiments with mice and dogs irradiated with LD50, it was shown the postirradiation depopulation of haemopoietic polypotent (CFUs) cell-precursors in mouse bone marrow was more pronounced than that of granulocytic and macrophagal cells (CFUdc). The rate of repopulation of CFUs during the first week was higher than that of CFUdc (T1/2 was 2.5 and 8.8 days respectively). In dogs, one could notice a partial change in the colony formation, a prolonged plateau period in the postirradiation CFUdc dynamics, and a coincidence in time with cellularity restoration in the bone marrow and peripheral blood leukocytes. It is suggested that in conditions of heterogeneous incubation in diffuse chambers, the haemopoietic cell-precursors are more mature than in the syngeneic system. The method of CFUdc determination has proved to be ineffective in estimating the onset and intensity of the postirradiation haemopoiesis recovery in dogs. The study of the bone marrow CFUdc population may, however, be used in intact animals to predict the probability of their death after irradiation within the median lethal dose range.     

Factors that control the differentiation of stem cells. I. The change in direction of hematopoietic stem cell differentiation under the effect of differentiating T-lymphocytes]

A mixed transplantation of bone marrow cells, and lymph nodes or thymic cells of mice CBA strain into lethally irradiated hybrid recipients (CBAXC57B1)F1 is accompanied with changes in the differentiation pattern from a mainly erythroid to a mainly granuloid way. Thymectomy of either donor of bone marrow cells or recipients, or both, destroys the stem cell differentiation in the direction of granulopoieseis. Intact syngeneic lymphocytes normalize differentiation of the stem cells, but in the presence of tissue antigens these provide for the stem cell differentiation mainly in the direction of granulopoiesis. The differentiation of stem haemopoietic cells is accomplished under the thymic and lymphocyte control. T-differentiating lymphocytes (Td) are the lymphocytes controlling the stem cell differentiation.     

Differential proliferation of erythroid and granuloid spleen colonies following sublethal irradiation of the bone marrow donor

Spleen colonies produced by sublethally irradiated mouse bone marrow cells were compared to those produced by unirradiated marrow cells in lethally irradiated mice. Sublethally irradiated marrow cells gave rise to many fewer spleen colonies. At seven days of colony age, the ratio of erythroid colonies to granuloid colonies was lower (< 1) than for colonies formed by unirradiated marrow (2 to 3 or more). Delay of harvest of colonies to day 10 or 12 resulted in 6 to 11 fold increase in the ratio of erythroid to granuloid colonies due largely to the belated appearance of erythroid colonies.     

The wall of the chick embryo aorta harbours M-CFC, G-CFC, GM-CFC and BFU-E

In the 3- to 4-day avian embryo, after the first wave of haemopoiesis which derives in the yolk sac from haemopoietic stem cells formed in situ, haemopoietic cells emerge in an intraembryonic site, the wall of the aorta. In this paper, we demonstrate that this site harbours M-CFC, G-CFC, GM-CFC and late and early BFU-E. In serum-free medium, the growth of M-CFC and GM-CFC was strictly dependent on CSF present in fibroblast-conditioned medium (FCM). The growth of G-CFC was improved when FCM was replaced by a minute quantity of chicken and fetal calf serum. Like erythroid progenitors from bone marrow, BFU-E detected here required anaemic chicken serum to differentiate into haemoglobinized cells. The frequency of the different types of haemopoietic progenitors in the aortic population was very high: 80 M-CFC, 25 G-CFC, 4 GM-CFC and 70 BFU-E for 12,500 aorta cells, i.e. two to eight times more frequent than in the bone marrow population, depending on the type of progenitors.     

Stem cell maintenance and commitment to differentiation in long-term cultures of murine marrow obtained from different spatial locations in the femur

Abstract. Murine bone marrow was separated into axial and marginal fractions in order to investigate the ability of cells from different spatial locations in the marrow to establish long-term cultures. The maintenance of haemopoiesis was significantly poor in long-term cultures of marginal marrow compared with axial or control (unfractionated marrow) cultures. Using techniques to further fractionate the axial or marginal marrow by depleting either stromal or haemopoietic cells, it was possible to investigate the relative importance of stromal and haemopoietic cell components. In the combinations studied, the more important determinant of effective in vitro haemopoiesis was the source of the haemopoietic cells rather than the stroma. The most effective stem cell maintenance and commitment to differentiation was observed when the source of the haemopoietic population was axial marrow. The data are consistent with axial marrow being a source of 'high quality' stem cells and this quality being an intrinsic property of the cells rather than one imposed by the stromal environment.     

Stem cell maintenance and commitment to differentiation in long-term cultures of murine marrow obtained from different spatial locations in the femur

Murine bone marrow was separated into axial and marginal fractions in order to investigate the ability of cells from different spatial locations in the marrow to establish long-term cultures. The maintenance of haemopoiesis was significantly poor in long-term cultures of marginal marrow compared with axial or control (unfractionated marrow) cultures. Using techniques to further fractionate the axial or marginal marrow by depleting either stromal or haemopoietic cells, it was possible to investigate the relative importance of stromal and haemopoietic cell components. In the combinations studied, the more important determinant of effective in vitro haemopoiesis was the source of the haemopoietic cells rather than the stroma. The most effective stem cell maintenance and commitment to differentiation was observed when the source of the haemopoietic population was axial marrow. The data are consistent with axial marrow being a source of 'high quality' stem cells and this quality being an intrinsic property of the cells rather than one imposed by the stromal environment.     

Estimates of the efficacy of the local protection of dogs against ionizing radiation

The possibility of prognosis of survival of dogs subjected to acute nonuniform irradiation leading to haemopoietic radiation sickness has been demonstrated. The prognosis is based on the "equal dose" concept that is implemented together with the dose dependence of the bone marrow haemopoiesis activity. The estimates of the consequences of irradiation of dogs, with certain body parts being shielded, have shown a good correlation between the calculation results and the experimental data.     

Presence of pluripotent haemopoietic precursors in vitro (CFU-mix) in haemopoietic tissues from mice of W/Wv genotype

Abstract. The presence or absence of haemopoietic precursors, which produce mixed colonies in vitro (CFU-mix) was examined in the bone marrow and spleen of (WB x C57BL/6) F1- W/W^v mice. Despite the failure of macroscopically evident colonyformation in the spleens of irradiated mice, haemopoietic cells of W/W^v mice did produce macroscopically-evident mixed colonies containing erythroid cells, macrophages, and often megakaryocytes, in culture medium. The size and constitution of mixed colonies derived from W/W^v mice were comparable to those of mixed colonies from congenic +/+ mice. The present results appear consistent with in vivo haemopoiesis in the W/W^v mice, which is obviously deficient, but sufficient for survival.     

胎肝造血干细胞的移植特性

胚胎发育中,肝脏是一个重要的造血器官。近年来胎肝移植的临床应用重新引起了人们的关注。本文应用染色体的 C-带染色法研究了小鼠骨髓和胎肝造血干细胞在照射受体小鼠中的增殖能力与相互间的竞争作用。实验结果表明胎肝造血干细胞在成年骨髓中的植入率比较同样条件下的成年骨髓造血干细胞低,但胎肝造血干细胞比较成年骨髓造血干细胞具有更强的自我更新或增殖能力。在同种胎肝造血干细胞移植中,为了降低同种移植抗力,提高移植的胎肝造血干细胞在受体中的耐受性,移植前对受体作适当的免疫抑制处理是必要的。因此,克服个体发育屏障和移植免疫屏障是提高同种胎肝造血干细胞移植效果中两个重要的研究课题。     

THE ROLE OF ERYTHROPOIETIN IN REGULATION OF POPULATION SIZE AND CELL CYCLING OF EARLY AND LATE ERYTHROID PRECURSORS IN MOUSE BONE MARROW

This study was designed to determine the stage in haemopoietic cell differentiation from multipotential stem cells at which erythropoietin becomes physiologically important. The responses of haemopoietic precursor cells were monitored in the bone marrow of mice under conditions of high (after bleeding) and low (after hypertransfusion) ambient erythropoietin levels. The number of relatively mature erythroid precursors (CFU-E), detected by erythroid colony formation after 2 days of culture, increased three-fold in marrow by the fourth day after bleeding, and decreased three-fold after hypertransfusion. Assessed by sensitivity to killing by a brief exposure to tritiated thymidine (³H-TdR) in vitro, the proliferative activity of CFU-E was high (75% kill) in untreated and bled animals, and was slightly lower (60% kill) after hypertransfusion. The responses of more primitive erythroid progenitors (BFU-E), detected by erythroid colony formation after 10 days in culture, presented a contrasting pattern. After hypertransfusion they increased slightly, while little change was noted until the fourth day after bleeding, when they decreased in the marrow. The same response pattern was observed for the progenitors (CFU-C) detected by granulocyte/macrophage colony formation in culture. The sensitivity of BFU-E to ³H-TdR was normally 30%, and neither increased after bleeding nor decreased after hypertransfusion. However, in regenerating marrow the ³H-TdR sensitivity of BFU-E increased to 63%, and this increase was not affected by hypertransfusion. These results are interpreted as indicating (1) that physiological levels of erythropoietin do not influence the decision by multipotential haemopoietic stem cells to differentiate along the erythroid pathway as opposed to the granulocyte/macrophage pathway; (2) that early erythroid-committed progenitors themselves do not respond to these levels of erythropoietin, but rather are subject to regulation by erythropoietin-independent mechanisms; and (3) that physiological regulation by erythropoietin commences in cells at a stage of maturation intermediate between BFU-E and CFU-E.     

Macrophages in haemopoietic and other tissues of the developing mouse detected by the monoclonal antibody F4/80.

Macrophages are widely distributed in lymphohaemopoietic and other tissues of the normal and diseased adult, where they play an important role in host defence and repair. Although the development of haemopoiesis has been well studied in several species, the ontogeny of the mononuclear phagocyte system remains poorly understood. We have used a highly specific mAb, F4/80, to examine the distribution of mature macrophages in the developing mouse, with special reference to their presence in the haemopoietic microenvironment. Monocytes and macrophages were first seen in embryos on day 10 in the yolk sac and liver as well as in mesenchyme. In liver, spleen and bone marrow, there was expansion of this population associated with the initiation of haemopoiesis on days 11, 15 and 17, respectively. Macrophages in these sites formed part of the haemopoietic stroma and their extensively spread plasma membrane processes could be seen making intimate contacts with clusters of differentiating haemopoietic cells. F4/80+ cells were widely dispersed in undifferentiated mesenchymal tissue in organs such as lung, kidney and gut. Numbers of F4/80-labelled cells increased concomitantly with organ growth and local mitoses were evident, as well as actively phagocytic macrophages. Our studies establish that macrophages are among the earliest haemopoietic cells to be produced during development and that they are relatively abundant in fetal tissues in the absence of overt inflammatory stimuli. Their distribution is correlated with the sequential migration of haemopoiesis and they constitute a prominent component of the stroma in fetal liver, spleen red pulp and bone marrow. Apart from a role in haemopoietic cellular interactions, their highly developed endocytic and biosynthetic activities suggest that macrophages contribute major undefined functions during growth, turnover and modelling of fetal tissues.     

Differential analysis of animal bone marrow by flow cytometry.

A simple procedure was developed for rapid analysis of animal bone marrow by flow cytometry using the lipophilic cationic dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)]. The batch process allows differentiation of bone marrow cells into lymphoid, erythroid, and myeloid populations and enables classification of erythroid and myeloid cells into proliferating and maturing subpopulations. From these data, myeloid:erythroid (M:E) ratios and maturation indices for erythroid and myeloid cells (EMI and MMI, respectively) can be derived. This procedure provides the opportunity to analyze bone marrow quantitatively and offers distinct advantages to current manual methods in terms of simplicity, throughput, and reproducibility. The method has been tested successfully using marrow from Wistar rats, B6C3F1 mice, beagle dogs, and cynomolgus monkeys. This technique facilitates the evaluation of bone marrow samples taken from preclinical safety studies or from animal colonies of large size.     

The kinetics of hematopoietic stem cells during and after hypoxia. A model analysis

A previously described mathematical model of the hematopoietic stem cell system has been extended to permit a detailed understanding of the data during and after hypoxia. The model includes stem cells, erythroid and granuloid progenitors and precursors. Concerning the intramedullary feedback mechanisms two basic assumptions are made: 1) The fraction "a" of CFU-S in active cell cycle is regulated. Reduced cell densities of CFU-S, progenitors or precursors lead to an accelerated stem cell cycling. Enlarged cell densities suppress cycling. 2) The self renewal probability "p" of CFU-S is also regulated. The normal steady state is described by p = 0.5, indicating that on statistical average each dividing mother stem cell is replaced by one daughter stem cell, while the second differentiates. Diminished cell densities of CFU-S or enlarged densities of progenitors and precursors induce a more intensive self renewal (p greater than 0.5), such that the stem cell number increases. The self renewal probability declines (p less than 0.5) if too many CFU-S or too few progenitors and precursors are present. The model reproduces bone marrow data for CFU-S, BFU-E, CFU-C, CFU-E, 59 Fe-uptake and nucleated cells in hypoxia and posthypoxia. Although the ratio of differentiation into the erythroid and granuloid cell lines is kept constant in the model, a changing ratio of CFU-E and CFU-C results. The model suggests that stem cells and progenitor cells are regulated by a regulatory interference of erythropoiesis and granulopoiesis.     

Hematopoietic differentiation cell surface antigen switching in the bone marrow of different aged chickens

Abstract. Immune cytolysis and immunofluorescence were used to examine chicken fetal antigen CFA) and chicken adult antigen (CAA) expression on the differentiation/maturation series of definitive erythroid cells obtained from the bone marrow of different aged chickens. We found that erythroid cells undergo changes in CFA/CAA antigenic expression dependent on their differentiation/maturation stage as well as the developmental age of the chicken. All differentiation/maturation stages of erythroid cells in the bone marrow of 12 and 18-day-old embryos express CFA only. Erythroblasts obtained from 7-day post-hatched chickens express either CFA or CAA. All three CFA/CAA phenotypes (i.e., CFA, CAA, and CFA + CAA) are observed in subsequent maturation stages, but only the CFA + CAA phenotype is observed in mature erythroid cells in the bone marrow of 7day post-hatched chickens. Erythroblasts from 62 day post-hatched chickens exhibit all three CFA/CAA phenotypes. Cells in the subsequent maturation stages express various CFA, CAA, or CFA + CAA phenotypes resulting in a majority of the mature erythrocytes expressing both CFA and CAA, and a small population of mature erythrocytes expressing CAA only. Erythroblasts from adult chickens express both CFA and CAA; however, CFA is lost during erythroid maturation resulting in mature erythrocytes which express CAA only. These studies indicate that both the erythroid differentiation/maturation stage and the developmental age of the chicken influence CFA and CAA antigenic expression on erythroid cells undergoing cellular differentiation/maturation in the bone marrow.     

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 (CFU_s) 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 CFU_s in the adult bone marrow culture, The results are discussed in the light of current concepts of haemopoietic stem cell regulation.     

Bone marrow stromal culture from patients with myelodysplastic syndromes and patients at different stages of acute nonlymphocytic leukaemia

The haematopoietic microenvironment or stroma plays a decisive role for the proliferation and differentiation of haemopoietic cells. We studied if bone marrow cells from patients with myelodysplastic syndromes (MDS) and acute nonlymphocytic leukaemias (ANLL) are altered in their ability to form adherent stromal layer with active haemopoiesis in the Dexter liquid culture. Bone marrow cells were obtained from 24 normal volunteers, 28 patients with ANLL in different stages of the disease and 9 patients with MDS. There are no differences between the stromal layers of patients with ANLL in complete remission and those of normal volunteers after two weeks of cultivation. However, bone marrow cells from patients with ANLL before treatment and from patients in relapse formed a poor adherent stromal layer in most cases. In 6 of 9 cases we found the normal stromal grade of bone marrow cells from patients with MDS. There were qualitative differences in the nonadherent cell population between normal and ANLL patients in complete remission. In most cases we found morphologically recognizable erythroid cells after two-weeks Dexter liquid culture of bone marrow cells from patients with ANLL in complete remission, which were not seen with normal volunteers. This could be an indication of harmful effects on the balance of haematopoiesis caused by previous infiltration with leukaemic cells or/and high-dose chemotherapy.     

Reduced variance of bone-marrow transit time of granulopoiesis—a possible pathomechanism of human cyclic neutropenia

Abstract. Human cyclic neutropenia (CN) is a haematological disorder characterized by oscillations in the numbers of neutrophilic granulocytes and other blood cells with a stable period of approximately 21 days. In most cases the neutrophils oscillate well below normal values such that these patients are chronically neutropenic. A comprehensive concept of the origin of CN is proposed. It assumes an abnormally small variance of the transit time of bone marrow cells (compared to normal human granulopoiesis) for the origin of the characteristic cycles. Furthermore, a reduced responsiveness of the immature granulopoietic bone marrow cells to the mitotic feedback stimuli is assumed to account for the subnormal neutrophil peaks. Together with feedback control provided in a simulation model of normal human granulopoiesis these two abnormalities can explain experimental and clinical cell kinetic data for bone marrow and blood in CN.     

Hematopoietic differentiation cell surface antigen switching in the bone marrow of different aged chickens

Immune cytolysis and immunofluorescence were used to examine chicken fetal antigen CFA) and chicken adult antigen (CAA) expression on the differentiation/maturation series of definitive erythroid cells obtained from the bone marrow of different aged chickens. We found that erythroid cells undergo changes in CFA/CAA antigenic expression dependent on their differentiation/maturation stages as well as the developmental age of the chicken. All differentiation/maturation stages of erythroid cells in the bone marrow of 12 and 18-day-old embryos express CFA only. Erythroblasts obtained from 7-day post-hatched chickens express either CFA or CAA. All three CFA/CAA phenotypes (i.e., CFA, CAA, and CFA + CAA) are observed in subsequent maturation stages, but only the CFA + CAA phenotype is observed in mature erythroid cells in the bone marrow of 7-day post-hatched chickens. Erythroblasts from 62 day post-hatched chickens exhibit all three CFA/CAA phenotypes. Cells in the subsequent maturation stages express various CFA, CAA, or CFA + CAA phenotypes resulting in a majority of the mature erythrocytes expressing both CFA and CAA, and a small population of mature erythrocytes expressing CAA only. Erythroblasts from adult chickens express both CFA and CAA; however, CFA is lost during erythroid maturation resulting in mature erythrocytes which express CAA only. These studies indicate that both the erythroid differentiation/ maturation stage and the developmental age of the chicken influence CFA and CAA antigenic expression on erythroid cells undergoing cellular differentiation/maturation in the bone marrow.     

Cellular interactions in erythroblastic islands in long-term bone marrow cultures, as studied by time-lapse video

Long-term bone marrow cultures (LTBMC) are readily converted from the usual granulopoietic to erythropoietic production by the addition of anemic mouse serum (AMS). The "statics" of proliferation and maturation, previously shown by ultrastructural methods to closely mirror the in vivo situation, were studied dynamically using a time-lapse video system. Several cell pedigrees were followed, but the most complete series showed three successive divisions and subsequent enucleations in the progeny of three synchronously mitotic cells observed in the culture; this is indicative of a five division sequence in the erythron. As in erythroblastic islets observed in marrow in vivo, the striking synchrony of maturation was maintained in vitro. Furthermore, when some of the erythroid progeny became displaced to other macrophages, the synchrony, which was maintained by the original erythroid group on the original erythroblastic islet macrophage, was lost. Time-lapse video, which is inexpensive to run and can be maintained in continuous recording for many weeks, is an ideal technique for recording both erythroid cell pedigrees, and the initial events leading to the formation of an erythroblastic islet in vitro after stimulation with AMS.