Pluripotent Stem Cells Do Not Completely Maintain Normal Human Steady-State Haemopoiesis

Since the classical work on the regulation of canine erythropoiesis by Alpen & Cranmore (1959), it has been generally accepted that recognizable bone-marrow cells are continuously replaced from sources of unrecognizable precursors. Although many features of pluripotent stem cells (PSC) and committed haemopoietic precursors have been determined, direct demonstration of a continuous influx, under normal steady-state conditions, from PSC into the recognizable bone-marrow cell compartments is still lacking. There is abundant evidence that PSC, in a number of species, including primates, resemble atypical or immature (‘transitional’) lumphocytes. By utilizing the technique of quantitative ¹⁴C-autoradiography, we have measured the activities of DNA and protein synthesis in individual bone-marrow cells of two healthy humans. A positive relationship was established between the protein synthesis rate and rate of movement through the cell cycle in all proliferative compartments. Lymphoid cells, considered to contain the fraction of PSC, were found in the lower range of this relationship. These low metabolic rates exclude fast growth as well as short cell-cycle times. In view of the low frequency of the potential PSC in the human bone marrow, amounting to less than 2%, these cells cannot be considered to represent a source continuously supplying the pool of rapidly proliferating, recognizable blast cells in the bone marrow under steady-state conditions. Some self-maintenance of a subcompartment within the pool of recognizable normal bone-marrow blast cells is therefore suggested.     

Granulocytopoiesis in children with acute lymphoblastic leukaemia.

Numbers, proliferative potential, and differentiative capacity of bone marrow granulocyte-macrophage precursor cells were studied in 130 children with acute lymphoblastic leukaemia (ALL), including 77 children in an acute phase of the disease and 53 in remission. Bone marrow samples from 65 children without haematopoietic abnormalities were used as controls. The numbers of clonogenic precursors were found to be below normal in all phases of ALL, particularly during the acute period when the bone marrow was heavily infiltrated with leukaemic cells. It is shown that the decreases in the numbers and proliferative potential of the precursor cells during the acute phases was associated with the effects of leukaemic blast cells, but that in remission the observed reduction in the precursor cell pool was due to the cytostatic effect of therapy. The differentiative capacity of clonogenic granulocyte and macrophage precursors was not altered in children with ALL.     

Prostaglandin E(2) and stem cell factor can deliver opposing signals to B lymphocyte precursors

The synthetic prostanoid, 16,16-dimethyl PGE(2), suppressed B lymphopoiesis in mice and proliferation of normal B cell precursors or the F10 pro-B cell line to interleukin 7 in culture. This was not the case with two other prostanoids, PGD(2) and PGF(2alpha), or agonists for PGI(2) agonist and thromboxane A(2) agonist receptors. PGE(2), but not the related prostanoids or agonists, induced apoptosis in F10 cells. The apoptotic response was mediated by the EP2 class of PGE(2) receptors and required an increase in intracellular cyclic adenosine 3',5'-monophosphate, activation of protein kinase A, and protein synthesis. The influence of PGE(2) on F10 cells was diminished in the presence of a cloned stromal cell line or stem cell factor. These findings describe another potential regulatory circuit in bone marrow which might influence B lymphopoiesis under disease or steady-state conditions.     

Interactions between lymphocytes and hematopoietic progenitor cells in normal and leukemic human bone marrow (phase contrast observations)

Single cell observations of normal and of leukemic human bone marrow cells demonstrated cell-cell interactions of lymphocytes with hematopoietic progenitor cells. In all cases lymphocytes and target cells were from the same individual. Lymphocyte-target cell interactions occurred more frequently with normal committed progenitor cells and leukemic blast cells from acute myeloid leukemia than with precursor cells of the proliferative cell pool, including myeloblasts, promonocytes, erythroblasts and megakaryocytes. Both induction of mitosis and degeneration of the progenitor cells occurred after cell-cell interaction with almost the same frequency. Acute myeloid leukemic blast cells degenerated after contact with lymphocytes with the same frequency as normal progenitor cells (i. e. in 16% of cell contacts), but especially during mitosis. In contrast, normal and regenerating bone marrow progenitor cells from myeloproliferative diseases demonstrated no degeneration after cell-cell interaction with lymphocytes during mitosis. Otherwise the induction of mitoses by lymphocyte-target cell interactions was more frequently observed in normal progenitor cells than in leukemic blasts.     

AN EARLY RESPONSE OF THE MORPHOLOGICALLY RECOGNIZABLE ERYTHROID PRECURSORS TO BLEEDING

⁵⁵Fe autoradiography of the peripheral red blood cells has been used to study the proliferation of the recognizable erythroid precursors in bled animals. The transit time of the recognizable erythroid precursors present in the bone marrow and labelled with ⁵⁵Fe 6 hr before bleeding, remains unchanged, but the number of red cells produced by these precursors is significantly greater than normal. It is deduced that the increased red cell production is brought about by an increase in the number of divisions that the cells undergo during maturation and that a shortening in the red cell cycle time is implied. The possibility that the transit time of the progeny of cells differentiating into pro-erythroblasts after bleeding may be shorter than the transit time of the precursors already differentiated before bleeding, is briefly discussed.     

AN ANALYSIS OF BONE MARROW ERYTHROPOIESIS IN THE MOUSE

Following the model of the erythropoietic system developed in the rat by Tarbutt and Blackett, the authors have carried out a kinetic analysis of bone marrow erythropoiesis in the mouse.
Using ⁵⁹Fe labelling techniques the size of the recognizable precursor cells and of the functional cell compartments have been estimated, while the flow-rate from the unrecognized precursor cells to the recognizable cells and from the latter compartment to the circulating erythrocytes have been evaluated by ⁵⁵Fe autoradiography.
Differences in the kinetic parameters of the erythropoietic mouse bone marrow compared with the rat bone marrow are reported, whose interpretation has required a more detailed analysis of the original model.     

Numbers and functions of transplantable primitive immunohematopoietic stem cells. Effects of age

This report introduces a new method in immunology, a use of the binomial formula with covariance to estimate numbers and proliferative patterns of the most primitive lymphoid precursors. We studied the primitive stem cells (PSC) from which most circulating lymphocytes and erythrocytes were descended during 300 to 400 days in recipients of genetically distinguishable marrow mixtures in competitive repopulation. Equivalent PSC concentrations (Eq. PSC Conc. or Conc.) were estimated, with the notion of common PSCs contributing equally in lymphoid and myeloid compartments. Similar estimation was done for common PSCs from which lymphocytes (and erythrocytes) drawn at successive sampling times about 100 days apart were descended. The percentages of lymphocyte and erythrocyte types, P1 and Pe, measured in each recipient were closely correlated, especially after 6 months and later. Close correlations were also found in cells sampled at successive one hundred day intervals, especially after the first. Apparently a few PSCs or their direct descendents produced most of the blood lymphocytes and E, and this production continued for many months. Concentrations of these PSCs (Equivalent PSC concentrations) were about one per 10(5) marrow cells from young donors. This is much lower than previous estimates, probably because our methods focus only on the most interesting precursors, those from which most of the circulating cells were descended. Equivalent PSC concentrations were about two-fold higher in old donors; old marrow produced correspondingly higher P1 and Pe values, but these declined with time. There were also small increases with time in the P1 and Pe values with young donors. To explain the temporal trends, we suggest that excess concentrations of precursors less primitive than PSC are present in old marrow, and their contribution to the differentiated cell population gradually declines. Possibly such precursors, as well as true PSC, proliferate in old donors to compensate for deficiencies that develop with age.     

Studies on the generation of B lymphocytes in fetal liver and bone marrow.

With the use of immunofluorescence techniques, cells containing cytoplasmic IgM (cIgM+), but lacking detectable surface IgM (sIgM+), have been identified in mouse fetal liver and adult bone marrow as a distinct cell population to sIgM+ B lymphocytes. We have shown that there is a considerable difference in the rate of entry of cIgM+ and sIgM+ cells into DNA synthesis in these locations. Moreover, within the cIgM+ population, the largest cells are the main group entering DNA synthesis. Our results are compatible with the notion that a pool of rapidly proliferating, large cIgM+ cells is present in fetal liver and adult bone marrow and that these cells give rise to populations of smaller cIgM+ cells, which move out of cell cycle, and convert to sIgM+ B lymphocytes. However, we recognize that this interpretation is speculative. Finally, we have shown that fetal bone marrow is a site of generation of sIgM+ B lymphocytes, but the question as to whether these cells are derived from Ig- precursors within marrow itself remains open.     

Bone marrow-thymus axis in senescence

This presentation offers a brief review of the bone marrow-thymus axis in senescence, a putative model for thymocyte differentiation, and recent results of our work on the status of pre-thymic stem cells in aged mice. The data presented here provide further evidence for a thymus endocrine influence on the bone marrow stem cells, specifically lymphocyte precursors. It has been postulated that the thymic hormones may act on lymphocyte precursors in the bone marrow and that the loss of thymic factors during senescence may be a contributing factor to the decreased cellular immune function. This study used Haar's in vitro model to investigate the bone marrow-thymus axis in aged mice. Erythroid-depleted bone-marrow cells from 3-month- and 24-month-old CBA (Thy 1.2) mice were placed in the upper half of a blind-well chamber with thymus supernatant in the lower half. Experimental cells were treated with thymus supernatant for 1 hr prior to migration. This study confirmed that pre-thymic stem cells in aged bone marrow are deficient in their ability to migrate to the thymus supernatant. It also revealed that treatment of the old bone marrow with thymus supernatant, made from neonatal thymus cultures, could dramatically improve the thymus migrating ability of the aged bone-marrow stem cells.     

In vitro study of bone marrow from leukemia patients and patients without hematologic disease

The bone-marrow of 26 patients not affected with hematological diseases and 10 patients with untreated leukemia was investigated according to Dexter in long-term cultures. Survival time and cell content of those long-term cultures started with normal bone marrow were not influenced significantly, if reinoculation was made with autologeneic or allogeneic bone marrow. Even without repeated inoculation, leukemic cells grew for a longer time in long-term cultures than normal bone marrow cells. As far as the outcome of the disease is concerned, no conclusion can be drawn from the duration of cultivating leukemic cells growth.     

Neutrophil marrow release. A system analysis.

Neutrophil marrow egress is governed by several processes. The most important are cell maturation, functional behavior of marrow sinusoids and humoral or neuro-vascular factors. Neutrophil release cannot be observed directly but is reflected in the size, cellular composition and kinetics of the nonproliferating pool of granulocytopoiesis in bone marrow and of blood neutrophil pool. These experimentally determined parameters were used as the basis of a mathematical model study. The model describes two catenated compartments, the nonproliferating pool of granulocytopoiesis in marrow and the total blood granulocyte pool. Cell transit from one pool to the other was assumed to be age-dependent. It was expressed by a positive sloping sigmoidal function that defines the egress potential fo the cells that increases with cell maturation. During maturation granulocytopoietic cells develop intense motility which determines the morphology of the cells on smears. Relationship between cell motility and its morphology was defined by functions determining the age-dependent probabilities of cell fixation as metamyelocytes, band- and segmented forms, respectively. The parameters of this model could be so adjusted that all experimental data were matched within experimental errors. Thus, qualitative and quantitative information on neutrophil marrow egress was obtained for normal and pathological states of granulocytopoiesis.     

Evidence for a common leukemia-associated antigen in acute myelogenous leukemia

Summary An antiserum was raised in rabbits to extracts of a pool of acute myelogenous leukemia cells. The immunization protocol used (antibody feedback) gave rise to antisera with marked specificity for AML extracts. After absorption, the antiserum demonstrated essentially no reactivity with cell extracts from 12 individual normal peripheral blood samples, while it reacted positively with 16 individual extracts from AML cells. Reactivity was assayed by the enzyme-linked immunosorbent assay (ELISA). The antiserum was not reactive with extracts from normal PHA-induced blast cells, with extracts of bone marrow cells from six individuals, or with three individual extracts of chronic lymphocytic leukemia (CLL) blast cells. These data indicate that this antiserum is detecting an antigen that is common to AML cells but may not be common to other blast cells.     

IgM rheumatoid factor autoantibody and immunoglobulin-producing precursor cells in the bone marrow of humans

The natures of the IgM rheumatoid factor (RF)-, IgM-, and IgG-secreting cells in the human bone marrow as compared to the peripheral blood, have been investigated by (1) response to the polyclonal B-cell activator, the Epstein-Barr virus (EBV), (2) sensitivity to the S-phase specific antimetabolite hydroxyurea, (3) presence of the BA-1 and Ia antigens on the cell surface, and (4) cell size, as determined by counter flow elutriation. The EBV-inducible bone marrow IgM-RF precursors derived from medium to large B cells that were inhibited by hydroxyurea pretreatment. The marrow total IgM response derived from small to medium size cells, and was only partially inhibited by hydroxyurea. Hydroxyurea had no effect on IgM-RF or IgM synthesis by peripheral blood cells. These results indicate that the marrow EBV-induced IgM-RF response is not representative of the response by peripheral blood cells, moreover; the marrow RF secreting response arises from a dividing cell pool that may represent newly generated autoreactive B cells.     

Isolation of two early B lymphocyte progenitors from mouse marrow: a committed pre-pre-B cell and a clonogenic Thy-1-lo hematopoietic stem cell

Two novel early B lymphocyte precursor populations have been identified by their capacity to differentiate in Whitlock-Witte bone marrow cultures. Cells expressing neither the B lineage antigen B220 nor Thy-1 contain committed B cell precursors which differentiate in short-term culture into pre-B and B cells. The other population expresses low levels of Thy-1, and lacks B220 as well as the T cell markers L3T4 and Lyt-2. The Thy-1+ cells which initiate long-term B cell cultures contain clonogenic B cell precursors at a frequency of 1 in 11, a 100-fold enrichment over unseparated bone marrow. Thy-1+ cells are also highly enriched for myeloid-erythroid precursors (CFU-S). Thy-1+ cells allow long-term survival of lethally irradiated mice and fully reconstitute the hematopoietic system, including T and B lymphocyte compartments. These results indicate that this population (approximately 0.1% of bone marrow) may contain the pluripotent hematopoietic stem cell.     

The effect of periodic starvation on bone-marrow cells from animals of various ages

At present, little is known of what happens to bone-marrow cells under caloric restriction or periodic starvation. The aim of the study was to investigate the influence of periodic starvation on the proliferative potential and cell morphology of the heterogeneous bone-marrow pool of young and old rats. Periodic starvation reduced the number of bone-marrow cells independently of the animal age, but did not affect cell viability and morphology. In young rats, periodic starvation increased the proportion of differentiated cells in the bone-marrow population; in old animals, it increased the number of undifferentiated blasts and blood-cell precursors. Bone-marrow cell cultivation was accompanied with reduced cell heterogeneity. Periodic starvation increased proliferative potential of bone-marrow cells from old rats. This suggests that periodic starvation at late stages of ontogenesis has an antiaging effect on bone-marrow cells in vivo and in vitro.     

Novel surface expression of reticulocalbin 1 on bone endothelial cells and human prostate cancer cells is regulated by TNF-alpha

An unbiased cDNA expression phage library derived from bone-marrow endothelial cells was used to identify novel surface adhesion molecules that might participate in metastasis. Herein we report that reticulocalbin 1 (RCN1) is a cell surface-associated protein on both endothelial (EC) and prostate cancer (PCa) cell lines. RCN1 is an H/KDEL protein with six EF-hand, calcium-binding motifs, found in the endoplasmic reticulum. Our data indicate that RCN1 also is expressed on the cell surface of several endothelial cell lines, including human dermal microvascular endothelial cells (HDMVECs), bone marrow endothelial cells (BMEC), and transformed human bone marrow endothelial cells (TrHBMEC). While RCN1 protein levels were highest in lysates from HDMVEC, this difference was not statistically significant compared BMEC and TrHBMEC. Given preferential adhesion of PCa to bone-marrow EC, these data suggest that RCN1 is unlikely to account for the preferential metastasis of PCa to bone. In addition, there was not a statistically significant difference in total RCN1 protein expression among the PCa cell lines. RCN1 also was expressed on the surface of several PCa cell lines, including those of the LNCaP human PCa progression model and the highly metastatic PC-3 cell line. Interestingly, RCN1 expression on the cell surface was upregulated by tumor necrosis factor alpha treatment of bone-marrow endothelial cells. Taken together, we show cell surface localization of RCN1 that has not been described previously for either PCa or BMEC and that the surface expression on BMEC is regulated by pro-inflammatory TNF-alpha.     

骨髓刺激后骨髓干细胞移植治疗下肢缺血的研究进展

自体骨髓干细胞移植治疗下肢缺血的研究是近年来令人关注的领域,取得一定的进展,但疗效有待进一步提高。骨髓刺激后自体骨髓干细胞移植治疗肢体缺血取得了较好的疗效;同时它具有抽取骨髓血少、获得细胞量多且安全性高的优点。但其具体作用机理尚不十分明确。内皮祖细胞数量的增加与质量的提高、局部微环境的改变可能是骨髓刺激后自体骨髓干细胞移植促进肢体缺血改善的机制。     

Contribution of bone-marrow-derived cells to choroidal neovascularization

We investigated the involvement of bone-marrow derived cells to experimental choroidal neovascularization (CNV) in mice, whose bone marrow was reconstituted by either unfractionated bone-marrow cells or Lin-c(-)Kit(+)Sca-1+ enriched presumable hematopoietic stem cells from the green fluorescent protein (GFP) transgeneic mice. Immunohistochemical analysis demonstrated the presence of GFP-positive cells in the CNV lesion after unfractionated bone-marrow transplantation, as well as Lin-c(-)Kit(+)Sca-1+ cell transplantation. Some of the GFP-expressing cells also expressed CD-31 and PanEC antigen, markers of vascular endothelial cells. Our results suggest that bone-marrow derived cells may contribute endothelial cells in CNV.     

The age-dependent loss of bone marrow B cell precursors in autoimmune NZ mice results from decreased mitotic activity, but not from inherent stromal cell defects

The formation of B lymphocytes is abnormal in autoimmune NZB and (NZB x NZW)F1 mice. With age, the proportion of sIg- Ly-5(220)+ pre-B cells and less mature B cell progenitors in the bone marrow progressively declines, reaching only approximately one-third of normal levels in 20-wk-old NZ mice. To determine the mechanisms responsible for the deficiency of NZ B lineage precursors, the mitotic activity of sIg- Ly-5(220)+ bone marrow cells in vivo was determined in NZ and conventional inbred mice as a function of age. The proportion of sIg- Ly-5(220)+ B cell precursors in (S + G2/M) stages of the cell cycle steadily decreased with age in NZ autoimmune mice. Furthermore, upon metaphase arrest, the rate of entry of sIg- Ly-5(220)+ bone marrow cells into G2/M also decreased with age in NZ mice. Therefore, the mitotic activity of sIg- Ly-5(220)+ B cell precursors is substantially decreased in NZ mice greater than or equal to 20 wk of age. The capacity of the bone marrow stromal microenvironment of NZ mice to support B lineage precursor growth was tested in two ways: 1) the capacity of preformed NZ bone marrow stroma to support B lineage cell growth in long term bone marrow cell culture under lymphopoietic conditions was assessed and 2) the capacity of NZ bone marrow B lineage precursors to expand in vivo after sublethal (200 rad) whole body irradiation was determined. Stroma derived from adult NZ mice supported the growth and development of B lineage lymphocytes in long term bone marrow cell culture to a greater extent than did age-matched conventional murine stroma. Furthermore, sublethal irradiation of older adult NZ mice resulted in some expansion of bone marrow sIg- Ly-5(220)+ B cell precursors in vivo. Therefore, the deficiency of B cell progenitors in the bone marrow of older NZ autoimmune mice is associated with diminished mitotic activity. However, this does not result from defects in the capacity of NZ bone marrow stroma to permit B lineage cell expansion as determined by both in vitro and in vivo experiments. In the absence of a detectable stromal cell defect, it is possible that an active inhibitory process within the bone marrow influences the mitotic activity of B cell precursors in NZ mice.     

A mathematical model of canine granulocytopoiesis

Summary The granulocyte cell renewal system of the dog is represented by a mathematical model consisting of the following compartments: The pool of pluripotential stem cells, the committed stem cell pool, divided into a blood and a bone marrow compartment, the proliferation pool, the maturation pool, the reserve pool and the blood pool of functional granulocytes. This chain of compartments is described by a system of non-linear differential equations. Cell losses anyplace in the system provoke increased production in all pools containing cells capable to divide. A reduced number of granulocytes in the blood pool stimulates production of a granulocyte releasing factor which mobilizes a rising number of cells to transit from the marrow reserve into the blood pool.The model was simulated on a digital computer. It was found to be capable to reproduce the steady state conditions and it also fits the data of two distinct experimental perturbations of the system both equally well. These perturbations are a loss of proliferating cells as it occurs after the administration of cytostatic drugs and losses of functional cells as they are induced by leukapheresis experiments of differing leukapheresis rates.This study was supported by the Deutsche Forschungsgemeinschaft (SFB 112)