Cells Forming Spleen Colonies At 7 Or 11 Days After Injection Have Different Proliferation Rates

Abstract In the early periods (7–9 days) after haemopoietic cell injection, colonies produced by CFU-s and by their progeny are identified in the spleen, while at later periods (11 days after injection) only spleen nodules produced by CFU-s persist. the increase in the suicide values of CFU-s after sublethal (2 Gy) irradiation of mice is associated with a higher proliferation rate of precursors of transitory spleen colonies, but not of CFU-s, as measured by different suicide techniques. During the log-phase of cell growth in a lethally irradiated recipient, the injected CFU-s and CFU-tr proliferate at a higher rate. Active proliferation of CFU-s and CFU-tr has been demonstrated in long-term bone-marrow cultures by the hydroxyurea in-vitro suicide assay. CFU-tr may be the cause of artifactual effects during measurement of haemopoietic stem-cell cycling by CFU-s suicide methods.     

Progenitors of transitory spleen colonies in mouse embryonal liver

The transitory nature of about half of spleen colonies macroscopically detectable in the spleen 7 days after injection of embryonal liver hemopoietic cells was demonstrated by localization of the colonies on the spleen surface and by the study of the content of polypotential and unipotential precursors in individual 7- and 11-day colonies produced in the spleen of irradiated mice by the cells of early (12-13-day) and late (17-18-day) embryonal liver.     

The cellular composition of hemopoietic spleen colonies

The cellular composition of individual hemopoietic spleen colonies has been studied using techniques which tested primarily for cell function rather than cell morphology. Erythroblastic cells were recognized by their capacity to incorporate radioiron, granulocytic cells by their content of peroxidase-positive material, and hemopoietic stem cells by their ability to form spleen colonies in irradiated hosts. It was found that, 14 days after the initiation of spleen colonies, the distribution of these cell types among individual colonies was very heterogeneous, but that most colonies contained detectable numbers of erythroblasts, granulocytes and colony-forming cells. An appreciable proportion of the cells in the colonies could not be identified as any of these three cell types. No strong correlations between numbers of erythroblasts, granulocytes and colony-forming cells in individual colonies were observed, though there was a tendency for colonies containing a high proportion of erythroblasts to contain a low proportion of granulocytes, and for colonies containing a high proportion of granulocytes to contain a higher proportion of colony-forming cells. An analysis of colonies which contained cells bearing radiation-induced chromosomal markers indicated that 83–98% of the dividing cells within 14-day spleen colonies were derived from single precursors.     

Effect of the cell components of Staphylococcus aureus on the functional activity of hemopoietic cells in mice

The data on the stimulating action of S. aureus cells, strains B-243, 2287, Wood-46, Cowan I, as well as cell-wall peptidoglycan, on the formation of endogenous colonies in the spleen of sublethally irradiated mice are presented. Teichoic acid, S. aureus ribosomal and cytoplasmic antigens produced no such effect. Whole S. aureus cells and their components were incapable of activating transitory colonies in the spleen of sublethally irradiated mice. After immunization with cell walls, peptidoglycan and protein A the mice showed a rise in the absolute and relative content of blood-forming stem cells in the marrow and the spleen. Killed S. aureus cells increased the relative content of blood-forming stem cells in the marrow, while in the spleen a rise in both absolute and relative content of such cells occurred, which was detected in the exocolonization test.     

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.     

Recovery of proliferative capacity of agar colony-forming cells and spleen colony-forming cells following ionizing radiation or vinblastine

The effects of sublethal radiation and the mitotic inhibitor, vinblastine sulphate, on the number of cells in mouse bone marrow capable upon transplantation of forming macroscopic colonies on the surface of the spleens of irradiated recipient mice (CFU) and on the number of cells capable of forming colonies in soft agar after cell culture (ACFU) were studied as a function of time after injury. The results show that ACFU are radiosensitive and vinblastine-sensitive cells, comparable in sensitivity to erythropoietin-sensitive cells. The temporal pattern of recovery following radiation of ACFU, different from that for CFU, is compatible with the concept that these are two distinct but closely related stem cell populations. The relevance of these findings to models of hematopoiesis and to studies on the precursors of macrophages and monocytes in inflammatory exudates is discussed.     

Gene analysis and dynamics of tumor stem cells in human glioblastoma cells after radiation

Glioblastoma is the most malignant central nervous system tumor. Patients with glioblastoma are treated with a combination of surgery, radiotherapy and chemotherapy; however, this effect is not satisfactory with regard to the prognosis. It is reported that the tumor stem cells affect recurrence, and radio- and chemotherapy resistance of the tumor, and that these cells play an important role in tumorigenesis and tumor progression. Using human glioblastoma cell lines (T98G and A172), irradiated (0, 30, 60 Gy) glioblastoma cells were prepared under the same conditions as clinical therapy. We analyzed cell proliferation rate, side population analysis by fluorescence-activated cell sorting and isolation of CD133⁺ cells, and performed genetic analysis (human stem cells) on these cells. We also investigated the difference in gene expression in the cells after radiation. The stem cell-related genes were highly expressed in the CD133⁺ cells compared with the CD133^? cells, suggesting that the cancer stem cells may be located in these CD133⁺ cells. In the T98G cell line, the cell proliferation rate of 30-Gy irradiated cells was higher than those of non-irradiated cells and 60-Gy irradiated cells. Stem cell-related genes were highly expressed in 30-Gy irradiated CD133⁺ T98G cells. In conclusion, we suggest that CD133⁺ cells may strongly affect tumor proliferation and the resistance against radiation therapy.     

Characterization of spontaneous bone marrow recovery after sublethal total body irradiation: importance of the osteoblastic/adipocytic balance

Many studies have already examined the hematopoietic recovery after irradiation but paid with very little attention to the bone marrow microenvironment. Nonetheless previous studies in a murine model of reversible radio-induced bone marrow aplasia have shown a significant increase in alkaline phosphatase activity (ALP) prior to hematopoietic regeneration. This increase in ALP activity was not due to cell proliferation but could be attributed to modifications of the properties of mesenchymal stem cells (MSC). We thus undertook a study to assess the kinetics of the evolution of MSC correlated to their hematopoietic supportive capacities in mice treated with sub lethal total body irradiation. In our study, colony-forming units-fibroblasts (CFU-Fs) assay showed a significant MSC rate increase in irradiated bone marrows. CFU-Fs colonies still possessed differentiation capacities of MSC but colonies from mice sacrificed 3 days after irradiation displayed high rates of ALP activity and a transient increase in osteoblastic markers expression while pparγ and neuropilin-1 decreased. Hematopoietic supportive capacities of CFU-Fs were also modified: as compared to controls, irradiated CFU-Fs significantly increased the proliferation rate of hematopoietic precursors and accelerated the differentiation toward the granulocytic lineage. Our data provide the first evidence of the key role exerted by the balance between osteoblasts and adipocytes in spontaneous bone marrow regeneration. First, (pre)osteoblast differentiation from MSC stimulated hematopoietic precursor's proliferation and granulopoietic regeneration. Then, in a second time (pre)osteoblasts progressively disappeared in favour of adipocytic cells which down regulated the proliferation and granulocytic differentiation and then contributed to a return to pre-irradiation conditions.     

Generation of murine stromal cell lines supporting hematopoietic stem cell proliferation by use of recombinant retrovirus vectors encoding simian virus 40 large T antigen.

The bone marrow is a complex microenvironment made up of multiple cell types which appears to play an important role in the maintenance of hematopoietic stem cell self-renewal and proliferation. We used murine long-term marrow cultures and a defective recombinant retrovirus vector containing the simian virus 40 large T antigen to immortalize marrow stromal cells which can support hematopoiesis in vitro for up to 5 weeks. Such cloned cell lines differentially supported stem cells which, when transplanted, allowed survival of lethally irradiated mice, formed hematopoietic spleen colonies in vivo, and stimulated lymphocyte proliferation in vitro. Molecular and functional analyses of these cell lines did not demonstrate the production of any growth factors known to support the proliferation of primitive hematopoietic stem cells. All cell lines examined produced macrophage colony-stimulating factor. The use of immortalizing retrovirus vectors may allow determination of unique cellular proteins important in hematopoietic stem cell proliferation by the systematic comparison of stromal cells derived from a variety of murine tissues.     

Hemopoietic precursor cell defects in nonanemic but stem cell-deficient W44/W44 mice

Hematopoietic stem cell deficiencies cause a severe macrocytic anemia in W/Wv mice. W44/W44 mice, on the other hand, are not anemic, but, since they accept marrow implants without prior total body irradiation, they have inherited a stem cell lesion. In an attempt to identify the aberrant stem cell(s), we have determined the concentration in W44/W44 marrow of hematopoietic precursors known to be deficient in W/Wv marrow. The in vitro erythroid burst-forming units (BFU-E), the in vivo spleen colony-forming units (CFU-S), and the cells that repopulate the erythroid compartment of stem cell-deficient mice were examined. The progenitors of 7-day bursts are dramatically reduced in W/Wv marrow but are present in normal concentrations in W44/W44 marrow. W44/W44 marrow CFU-S, unlike W/Wv, generate visible spleen colonies 10 days after injection into lethally irradiated recipients. The colonies are, however, smaller and at least 2 times less numerous than those produced from equivalent numbers of +/+ marrow. An additional defect was the inability of W44/W44 stem cells to compete with genetically marked +/+ cells during erythroid repopulation. An estimate of the number of W44/W44 stem cells needed to compete with +/+ cells was provided by enriching W44/W44 progenitors fivefold. Twice as many enriched W44/W44 marrow cells as unfractionated +/+ cells were required to replace competitor cells. This suggests that there are up to 10 times fewer stem cells somewhere in the W44/W44 erythrogenerative pathway. The data support the conclusion that an erythroid progenitor less mature than the BFU-E is one of the cells most severely affected by expression of the mutant gene.     

Comparative studies on the characteristics of proliferation and differentiation of spleen colony-forming cells

Using a single spleen colony transplantation technique and sex chromosome typing as a natural cytogenetic marker, most spleen colony-forming cells (CFC) in adult bone marrow or fetal livers of inbred LACA or C57 mice re-established hemopoiesis in lethally irradiated mice when the spleen colonies were sampled at 13 days after transplantation. However, most of the spleen colony-forming cells in the peripheral blood of normal mice possess little potential for proliferation and are less efficient in the re-establishment of hemopoiesis in lethally irradiated mice. The CFC population is heterogeneous in the mice. From the subsequent retransplantation of colonies from colony-forming cells in the peripheral blood, the simple assessment of spleen colony-forming units (CFU-s) content, based on the number of splenic colonies, does not reliably represent the content of hemopoietic stem cells.     

CELL PROLIFERATION IN HAEMATOPOIETIC SPLEEN COLONIES OF MICE: DIFFERENCE BETWEEN COLONIES DERIVED FROM INJECTED ADULT BONE MARROW AND FOETAL LIVER CELLS

Cell proliferation in mouse spleen colonies, derived from injected foetal liver and young adult bone marrow, was studied by measuring incorporation of radio-iodine-labelled 5-iodo-2'-deoxyuridine (IUdR). Foetal liver-derived colonies incorporated significantly more IUdR than marrow-derived colonies on the 8th and 12th days after cell injection. The data are consistent with the view that foetal haematopoietic stem cells are capable, on average, of producing larger descendant populations than are stem cells from young adults.     

Effects of226Ra and X-irradiation on the proliferative and differentiative ability of mouse hemopoietic stem cells

Summary The ability of hemopoietic stem cells to repopulate spleens of heavily irradiated syngeneic hosts in form of macroscopically visible clonal colonies of differentiating cells was studied in mice exposed for 32 and 4 weeks to internally deposited²²⁶Ra (0.56 and 0.46 µCi per mouse respectively) or to 100 rad X-irradiation. Exocolonizing test and cytological techniques were used for quantitative evaluation. The size of stem cell compartment was reduced and the function of the surviving stem cells was altered by radium and X-ray irradiation. The proliferation and maintenance of hemopoietic cell populations were found to depend not only on the numbers of stem cells but also on their multiplicative and differentiative capability.     

Permissive role of interleukin 3 (IL-3) in proliferation and differentiation of multipotential hemopoietic progenitors in culture

We studied the effects of interleukin-3 (IL-3) on colony formation by hemopoietic progenitors in methylcellulose cultures of spleen cells from 5-fluorouracil (FU)-treated mice. Purified IL-3 supported the growth of various types of multilineage colonies including blast cell colonies. The types of colonies were similar to those supported by pokeweed-mitogen spleen cell conditioned medium (PWM-SCM), except that IL-3 supported eosinophil and neutrophil expression better. Delayed addition of IL-3 to cultures 7 days after cell plating decreased the number of colonies to one-half the number in cultures with IL-3 added on day 0. It did not alter the proliferative and differentiation characteristics of late emerging multipotential blast cell colonies. These observations suggest that IL-3 does not trigger hemopoietic progenitors into active cell proliferation but is necessary for their continued proliferation. This permissive role of IL-3 is consistent with a stochastic model of stem cell proliferation which features random entry into cell cycle. IL-3 also supported the growth of multilineage colonies from single cells isolated from blast cell colonies by micromanipulation. This result shows that IL-3 acts directly on multipotential progenitors. Analysis of colonies derived from paired progenitors revealed disparate lineage expression and was in accordance with the stochastic model of stem cell differentiation.     

Physical separation of hemopoietic stem cells from cells forming colonies in culture

Mouse bone marrow cells in suspension were separated into a number of fractions on the basis of cell density by equilibrium density gradient centrifugation, or on the basis of cell size by velocity sedimentation. After each type of separation, the cells from the various fractions were assayed for their ability to form macroscopic spleen colonies in irradiated recipient mice, and for their ability to form colonies in a cell culture system. The results from either separation technique demonstrate that cells in some fractions formed more colonies in vivo than in the culture system, while cells in other fractions formed more colonies in culture than in the spleen. The results of control experiments indicate that this separation of the two types of colony-forming cells was not an artifact of the separation procedures. From these experiments it was concluded that the population of cells which form colonies in culture under the conditions used is not identical to the population of cells detected by the spleen colony assay.     

CFU-S and haematopoietic microenvironment in mice after fractionated irradiation. A study on spleen colonies.

The paper is aimed at evaluating the quantity and quality of the haematopoietic stem cells, CFU-S, in the bone marrow and the functional effectiveness of the haematopoietic microenvironment of the spleen in two time intervals after repeated exposure of mice to doses of 0.5 Gy gamma-rays once a week (total doses of 12 and 24 Gy). After irradiation, bone marrow was cross-transplanted between fractionatedly irradiated and control mice. The parameter evaluated were numbers of spleen colonies classified into size categories. The data obtained provide evidence for a significant damage to the CFU-S, concerning both their number and proliferation ability, after both total doses used. The functional effectiveness of the haematopoietic microenvironment of the spleen was impaired only in bone marrow recipients receiving a transplant after having been exposed to a total dose of 24 Gy; this dose combined with subsequent pre-transplantation irradiation resulted in a marked suppression of cell production within the spleen colonies formed from a normal bone marrow on the spleens of fractionatedly irradiated mice.     

Characterization of early B lymphocyte precursors present in long-term bone marrow cultures

Lymphoid precursor cells are present in long-term bone marrow cultures (LTBMC), but their differentiation into mature lymphocytes is blocked. A quantitative assay for B cell precursors in LTBMC, which gives a linear relationship between the number of grafted LTBMC cells and the frequency of B cell colony forming units (CFU-B) in the spleen and bone marrow of immunodeficient CBA/N mice 19 days after reconstitution, is described. Characterization of the B cell precursor indicates that this assay is detecting a very early precursor and not a B lymphocyte or a late pre-B cell. This conclusion is based on the observations that a) pre-B cells transformable by Abelson murine leukemia virus are not present in LTBMC by 3 days postrecharge and CFU-B are absent by 6 days postrecharge; b) late B cell progenitors capable of rapid repopulation of irradiated CBA/N mice are not present in LTBMC, since a lag in the kinetics of B cell reconstitution in animals grafted with LTBMC cells is observed compared with fresh bone marrow cells; c) the B cell precursors in LTBMC have high proliferative potential, since they can stably repopulate recipient mice for at least 8 wk postreconstitution and through two serial passages in irradiated CBA/N recipients; and d) the B cell precursors are large, rapidly sedimenting cells as determined by velocity sedimentation. The serial transplantation experiment further shows that a split is often observed between lymphoid and myeloid reconstituting ability of LTBMC cells. The LTBMC B cell precursor may be a pluripotent stem cell or a lymphoid stem cell, although its differentiative potential remains to be determined.     

Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers

Abstract. Fibroblast colonies (clones) were obtained by explantation of bone marrow single-cell suspensions and were used to establish multicolony and single-colony derived fibroblast cultures by successive passaging of either pooled or individual colonies. When transplanted in diffusion chambers after 20–30 cell doublings in vitro , the descendants of fibroblast colony-forming cells (FCFC), whether grown from single or pooled colonies, retained the ability for bone and cartilage formation. The content of osteogenic precursors in the cultured progeny significantly outnumbered the initiating FCFC. Thus the high proliferative potential of bone marrow FCFC and their ability to serve as common precursors of bone and cartilage-forming cells makes them probable candidates for the role of osteogenic stem cells.