Comparison of stem-cell recovery in autoimmune and normal strains

The capacity of NZB stem cells to proliferate in vivo was evaluated in two systems which required repopulation of peripheral organs. In both types of depletion systems, stem-cell repopulation after cyclophosphamide treatment or adoptive transfer repopulation in lethally irradiated hosts, it was found that NZB stem cells were hyperproliferating. The increase in proliferating cells was most pronounced in the spleens of NZB mice treated with high-dose cyclophosphamide and in lethally irradiated F₁ mice reconstituted with NZB T-cell-depleted bone marrow. Thus, upon a stimulus to repopulate, NZB marrow stem cells will hyperproliferate in peripheral organs resulting in an increase in cell number. The abnormality in the marrow cells can be observed in young NZB mice when their marrow cells are in an environment which requires recovery and division.     

Dose- and time-related quantitative and qualitative alterations in the granulocyte/macrophage progenitor cell (GM-CFC) compartment of dogs after total-body irradiation

The effects of single-dose total-body X irradiation (TBI) on the granulocyte/macrophage progenitor cell (GM-CFC) population in bone marrow and blood of dogs were studied for dose levels of 0.78 and 1.57 Gy up to 164 days after irradiation. The blood GM-CFC concentration per milliliter was depressed in the first 7 days in a dose-dependent fashion to 5-16% of normal after 0.78 Gy and to between 0.7 and 5% after 1.57 Gy. The bone marrow GM-CFC concentration per 10(5) mononuclear cells, on the other hand, was initially reduced to about 45% of the average pre-irradiation value after 0.78 Gy and to 23% after 1.57 Gy. The regeneration within the first 30 to 40 days after TBI of the blood granulocyte values and the repopulation of the bone marrow GM-CFC compartment was associated with both a dose-dependent increase in the S-phase fraction of the bone marrow GM-CFC and a dose-dependent increase in colony-stimulating activity (CSA) in the serum. The slow repopulation of circulating blood GM-CFC to about only 50% of normal even between days 157 and 164 after TBI could be related to a correspondingly delayed reconstitution of the mobilizable GM-CFC subpopulation in the bone marrow.     

On the origin of hematopoietic stem cells after local marrow extirpation.

The early hematopoietic regeneration in a depopulated segment of femur shaft is compared in +/+ and W/Wv mice and in W/Wv mice previously treated with +/+ marrow. Since the W/Wv mouse has an intrinsic CFU deficiency on spleen colony assay and since immigrant cells play a negligible role in the onset of regeneration after marrow extirpation, the W/Wv(+/+) chimera provides a model for evaluation of the contribution of residual cells to the regenerative program. There was little difference in the relative recovery of CFU in +/+, W/Wv, and W/Wv-(+/+), Moreover, +/+ derived CFU were responsible for nearly all of the CFU repopulation in chimeric mice. Thus, recovery of hemic cellularity must be due to residual stem cells rather than to stem cells derived by transformation of more primitive mesenchymal elements. The residual CFU are probably intimately associated with bone, most likely within the endosteum and haversian system.     

GROWTH OF STEM CELL CONCENTRATES IN DIFFUSION CHAMBERS (DC)

The growth in diffusion chamber (DC) of normal murine marrow and marrow separated by discontinuous albumin centrifugation was studied. The colony-forming cells (CFC) assayed in soft agar, total cell counts and differentials were measured in the DC over a 19 day period after intraperitoneal implantation into CF₁ mice. Growth of implants of normal marrow or fraction 3 (F3) in which CFC had been concentrated 1.7–3.9-fold were compared at an initial cell concentration of 1 × 10⁵. There was a good correlation between the number of CFC implanted with granulocyte production but not with macrophage production. When higher cell concentrations of normal unfractionated marrow were implanted growth was reduced as was recovery of CFC. In two experiments in which both CFU and CFC concentrations were measured there was a general correlation between the two.     

Migration of bone marrow cells to the thymus in mice after a single sublethal-dose irradiation

In sublethally irradiated mice, thymus repopulation is due first to the proliferation of surviving thymocytes followed by the multiplication of bone marrow derived prothymocytes. The migration of bone marrow cells to the thymus after a single sublethal whole-body X irradiation was studied by using fluorescein isothiocyanate as a cell marker. Irradiation increases the permissiveness of the thymus to the immigration of bone marrow cells. Furthermore, the post-Rx regenerating bone marrow cells exhibit migration capacities greater than the normal ones. The radiation induced changes in the bone marrow thymus interaction might play an important role in thymus regeneration after sublethal irradiation.     

Heterogeneity within the hematopoietic stem cell compartment: evidence for a marrow-seeding stem cell distinct from CFU-s

Using a chromosome marker within a syngeneic system, we investigated the seeding characteristics of murine hematopoietic stem cells after transplantation to irradiated hosts. The chromosome-marked test cells were allowed to compete with normal marrow cells in repopulating the spleen and marrow of irradiated mice. Although the seeding behavior of normal marrow could be predicted from the number of colony-forming units-spleen (CFU-s) transplanted, the marrow seeding of melphalan-treated marrow was 7-fold greater than expected. Repopulation of marrow by spleen cells was less effective than expected from the CFU-s content, while the reverse was true after repopulation by fetal liver cells. These differences were emphasized after treatment of cell donors with melphalan. The results were due primarily to differences in the lodging properties of the transplanted cells, those seeding in the marrow were less sensitive to melphalan than CFU-s. In some instances marrow-repopulating ability could be separated from peak CFU-s activity on a density gradient, suggesting a marrow-repopulating cell exists that is distinct from CFU-s.     

Expression of bone matrix proteins during dexamethasone-induced mineralization of human bone marrow stromal cells

Glucocorticoids have been shown to induce the differentiation of bone marrow stromal osteoprogenitor cells into osteoblasts and the mineralization of the matrix. Since the expression of bone matrix proteins is closely related to the differentiation status of osteoblasts and because matrix proteins may play important roles in the mineralization process, we investigated the effects of dexamethasone (Dex) on the expression of bone matrix proteins in cultured normal human bone marrow stromal cells (HBMSC). Treatment of HBMSC with Dex for 23 days resulted in a significant increase in alkaline phosphatase activity with maximum values attained on day 20 at which time the cell matrix was mineralized. Northern blot analysis revealed an increase in the steady-state mRNA level of alkaline phosphatase over 4 weeks of Dex exposure period. The observed increase in the alkaline phosphatase mRNA was effective at a Dex concentration as low as 10⁻¹⁰ M with maximum values achieved at 10⁻⁸ M. In contrast, Dex decreased the steady-state mRNA levels of both bone sialoprotein (BSP) and osteopontin (OPN) over a 4 week observation period when compared to the corresponding control values. The relative BSP and OPN mRNA levels among the Dex treated cultures, however, showed a steady increase after more than 1 week exposure. The expression of osteocalcin mRNA which was decreased after 1 day Dex exposure was undetectable 4 days later. Neither control nor Dex-treated HBMSC secreted osteocalcin into the conditioned media in the absence of 1,25(OH)₂D₃ during a 25-day observation period. The accumulated data indicate that Dex has profound and varied effects on the expression of matrix proteins produced by human bone marrow stromal cells. With the induced increment in alkaline phosphatase correlating with the mineralization effects of Dex, the observed concomitant decrease in osteopontin and bone sialoprotein mRNA levels and the associated decline of osteocalcin are consistent with the hypothesis that the regulation of the expression of these highly negatively charged proteins is essential in order to maximize the Dex-induced mineralization process conditioned by normal human bone marrow stromal osteoprogenitor cells. © 1996 Wiley-Liss, Inc.     

REPOPULATION OF THE BONE-MARROW STEM-CELL COMPARTMENT AFTER IRRADIATION*

Bone-marrow repopulation has been followed as a function of time after a single whole-body dose of 150 rads X-rays to (C3H × C57B1)F₁ hybrid mice. The number of nucleated cells per bone shaft has been estimated by direct counting and the number of progenitor cells by the exogenous spleen-colony assay method. Vinblastine was also administered under various schedules of time and dose in an endeavour to distinguish between cycling and dormant cells in the progenitor pool. The depopulation of total marrow cells induced by Vinblastine proceeds at a comparable rate in irradiated and in normal mice. On the other hand, the depopulation of the colony-formers is faster in irradiated than in normal animals. The data are interpreted to show that the fraction of cycling progenitor cells is larger in a haemopoietic tissue undergoing numerical expansion.     

Two classes of primitive pluripotent hemopoietic progenitor cells: separation by adherence

Methylcellulose cultures containing mouse marrow cells at low densities and partially purified preparations of erythropoietin and Interleukin-3 were scored after 2 weeks for the presence of macroscopic multilineage colonies (from "primary" CFU-macro GEMM). Whole cultures were then harvested and replanted to assess the number of "secondary" CFU-macro GEMM produced, but not detected, during the primary culture period. In such experiments adherent marrow cells yielded significantly higher numbers of secondary CFU-macro GEMM than did either fresh or nonadherent marrow cells. Removal of macroscopic colonies prior to replating showed that most secondary CFU-macro GEMM were not derived from primary CFU-macro GEMM. In vivo studies also revealed a differential effect of adherence separation on the frequency of day 10 CFU-S, which decreased, by comparison to cells capable of long-term repopulation, which increased. Primary adherent CFU-macro GEMM from 5-fluorouracil (5-FU) treated mice showed an 18-fold higher self-renewal capacity than their counterparts in normal marrow. Nevertheless the majority of secondary CFU-macro GEMM obtained from primary cultures of adherent 5-FU cells were again not derived from primary CFU-macro GEMM. Cells capable of immediately generating large multilineage colonies thus appear to represent an intermediate compartment of pluripotent progenitors whose self-renewal properties, may, however, vary over a considerable range. Our results further suggest that these progenitors are derived ultimately from a more primitive adherent cell whose tendency to begin to divide in vitro is low and whose presence correlates with cells capable of long-term myeloid repopulation in vivo.     

Early endotoxin tolerance is associated with alterations in bone marrow-derived macrophage precursor pools

Early endotoxin tolerance has been defined as the transient period after an initial sublethal exposure to LPS during which a normally responsive individual is rendered hyporesponsive. Little is known about the cellular mechanisms that underlie this phenomenon. In this study, an early tolerance system was established by the injection of mice with 25 micrograms of E. coli K235 LPS. Maximal hyporesponsiveness in response to a challenge injection was observed 3 to 4 days after the initial injection, and normal responsiveness returned by 8 days after the initial exposure to LPS. Further experiments described herein demonstrate that the acquisition and maintenance of the tolerant state coincides temporally with an increase in the number of macrophage progenitor cells in the bone marrow. Cell-sizing profiles of the bone marrow cells from tolerized mice indicate an enrichment for a population of cells that are significantly larger than in bone marrow preparations from control mice. By density gradient sedimentation, it was shown that the denser population of cells from tolerized mice contained the increased numbers of progenitor cells, which, by cytology, were immature monocytic cell types. The increased numbers of macrophage progenitors was sustained after a second (challenge) injection of LPS. These results indicate that early endotoxin tolerance is associated with an increase in a population of bone marrow cells that is enriched for macrophage progenitors and suggests the possibility that the lack of responsiveness observed during the hyporesponsive period is related to a failure of these immature cell types to respond to LPS.     

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.     

Failure of bone marrow cells to reconstitute T cell immunity in graft-vs-host mice

A chronic GVH reaction (detected by T cell immune deficiency) was induced in unirradiated, adult (C57BL/10 X B10.A)F1 mice by injecting them i.v. with 3 X 10(7) B10.A parental spleen cells. Thirty-four days later, attempts were made to reconstitute the GVH immune-deficient mice by whole-body irradiation and repopulation with bone marrow cells from normal syngeneic F1 mice. The reconstituted mice were tested for CTL responses 147 and 272 days after repopulation with normal F1 bone marrow. These GVH/chimera mice remained immunoincompetent for at least 272 days for CTL responses to hapten-self and H-2 allogeneic antigens.     

Regulation of bone marrow lymphocyte production: III. Increased production of B and non-B lymphocytes after administering systemic antigens

To examine the influence of exogenous stimuli on the genesis of lymphocytes in mouse bone marrow, the production rate and subsets of marrow lymphocytes were examined after a systemic injection of sheep red blood cells (SRBC). Radioautographic analysis after either pulse labeling or infusion of [³H]thymidine revealed a pronounced increase in the number of newly formed small lymphocytes appearing in the marrow, maximal 4–5 days after SRBC injection and dose related. The resulting expansion of the marrow lymphocyte population included both immature B cells and null cells, as shown by cell surface and cytoplasmic markers. Similar stimulation of marrow lymphocyte production followed an injection of either bovine serum albumin or mineral oil. No comparable stimulation occurred in either the thymus or the spleen. The results demonstrate that antigens and nonspecific irritants can exert a central effect in the bone marrow, producing a surge in the production of both primary B and non-B lymphocytes. The possible role of external stimulants in determining the normal rate of bone marrow lymphocyte production is discussed.     

Induction of apoptosis in bone marrow cells after treatment of mice with WR-2721 and gamma-rays: relationship to the cell cycle

Apoptosis and cell proliferation are accepted to be responsible for the maintenance of homeostasis in the hematopoietic system. Understanding of the mechanisms of action of the aminothiols and ionizing radiation on normal hematopoietic cells requires determination of the correlation between apoptotic cell death and cell cycle distribution. The effects of WR-2721 ((S)-2-/3-aminopropylamino/ethylphosphorothioic acid; Amifostine) and ⁶⁰Co gamma-rays on apoptosis and cell cycle progression in the mouse bone marrow were determined. Adult male Swiss mice were exposed to 6 Gy gamma-rays only, or pretreated with WR-2721, at a dose of 400 mg/kg body weight, 30 min before gamma-irradiation. The laser scanning cytometry APO-BRDU^TM assay based on simultaneous analysis of cellular DNA content and the in situ detection of DNA strand breaks was used to identify apoptotic cells and to reveal the cell cycle position of apoptotic and nonapoptotic cells. Temporary changes in the frequency of apoptotic cells with fluorescein isothiocyanate (FITC) labeling of DNA strand breaks, and all bone marrow cells including apoptotic and nonapoptotic ones, whose DNA stained with propidium iodide, were observed in the particular phases of the cell cycle throughout the 96-h period after WR-2721 application and gamma-irradiation. The cell cycle phase specificity of WR-2721 and ⁶⁰Co gamma-irradiation was shown in terms of induction of apoptosis in bone marrow cells. The patterns of alterations in the frequency of apoptotic cells and all bone marrow cells with respect to their cell cycle position were dependent on the agent(s) applied and the time interval after treatment of mice with WR-2721 and/or gamma-rays. A modulatory, suppressive action of WR-2721 on apoptosis induction and the cell cycle perturbation caused in normal cells of the mouse bone marrow by gamma-rays was found.     

A peptide from Testudo horsfieldii tortoise spleen as a potential helper for reducing acute radiation syndrome

The Middle Asian tortoise Testudo horsfieldii is one of the most radioresistant animals, with Lethal Dose (LD) 50/30 around 500 Gy. Extracts were prepared from different organs of the tortoise, and their biological activity was evaluated. Crude extract from the spleen was found to significantly increase survival of mice treated with lethal doses of radiation. In an iterative process, the active extract was purified by chromatography, and the fractions were screened for biological activity. Various vital parameters were monitored: peripheral blood leukocytes, spleen colonies, mitosis in the bone marrow, and survival after 30 days. The process concluded with the isolation, characterization, and synthesis of the tetrapeptide FTGN, which accelerated repopulation of the irradiated bone marrow at very low concentrations both in vivo and ex vivo. A fluorescently labeled derivative of the peptide was found to selectively associate to CD34⁺ stem cells, suggesting that the peptide mediates their proliferation and allows fast repopulation of hematopoietic lineages. Interestingly, the peptide protected animals from alopecia. The studies in experimental animals suggest that treatment with FTGN can potentially benefit patients who suffer bone marrow damage due to radiotherapy or chemotherapy and patients undergoing autologous or allogenic bone marrow transplantation.     

Growth regulation of the interstitial cell population in hydra. II. A new mechanism for the homeostatic recovery of reduced interstitial cell populations

The interstitial cells of hydra comprise a stem cell population, producing at least two classes of terminally differentiated cell types, nerve cells and nematocytes. Exposure to hydroxyurea (HU) results in selective depletion of interstitial cells from the tissue. The surviving cells subsequently recovered to normal levels, and the mechanisms involved in this repopulation were examined. Hydra were treated for varying times with HU such that interstitial cell numbers were reduced to 7 or 35% of normal. Subsequent growth of the epithelial and interstitial cell populations in these animals was monitored. The results indicate that the growth rates of these two cell types were only slightly different from untreated controls during the 4 weeks after HU exposure, implying that repopulation should not have occurred. However, recovery of the interstitial cell population was observed. Further analysis revealed that the interstitial cells in HU animals, unlike normal hydra, were not uniformly distributed in the body column, and were especially reduced in the budding region. In normal animals a constant fraction of the interstitial and epithelial cells are lost into buds. However, as a consequence of this nonuniform distribution a smaller fraction of the interstitial cells are displaced into HU buds, thereby retaining a higher proportion in the adult tissue. Calculations indicate that this mechanism of increased retention is of sufficient magnitude to account for 40-60% of the observed recovery after HU treatment.     

Remission of acute myeloid leukemia: studies of in vitro colony formation by bone marrow cells

Human bone marrow contains cells which form leukocyte colonies in semisolid culture media. Each leukocyte colony arises from a single colony-forming cell which is thought to be a unipotential stem cell, and which is subject to regulation in vitro by colony-stimulating factor. In acute myelogenous leukemia variable abnormalities in colony formation by marrow cells occur. Usually colony formation either fails to occur or the colonies that are formed are small and contain fewer than 50 cells. Similar abnormalities have been described in bone marrow dysfunction preceding overt leukemia. Usually remission of leukemia is accompanied by improved cloning by marrow cells. In this study three patients are reported in whom remission was associated with impaired cloning, and one of these patients has remained in continuous remission for a further 18 months. These observations suggest that remission status is not necessarily associated with repopulation of the bone marrow by normal hematopoietic cells.     

Cellular events during radiation-induced thymic leukemogenesis in mice: abnormal T cell differentiation in the thymus and defect of thymocyte precursors in the bone marrow after split-dose irradiation

Cellular events during the development of thymic lymphomas in young B10.BR mice given leukemogenic split-dose irradiation were studied by examining the differentiation of functional T lymphocyte precursors in the regenerating thymus. It was found that leukemogenic radiation treatment resulted in a sustained depression of the level of thymic cytotoxic T lymphocyte precursors (CTLp) and of mixed lymphocyte reactivity of thymus cells when assessed between 1 and 4 mo after irradiation, in spite of the fact that the total number of thymocytes was restored to the normal level within 2 mo and continued to increase thereafter. In vitro mixing studies of normal thymocytes with thymus cells from split-dose irradiated mice provided no evidence for active suppression as a mechanism for this depressed activity. The ability of bone marrow cells from split-dose irradiated mice to regenerate the thymus and to differentiate into functional CTLp was examined by use of supralethally irradiated Thy-1 congenic recipients. Reconstitution of supralethally irradiated B10.BR Thy-1.2 mice with normal bone marrow from B10.BR Thy-1.1 mice resulted in the complete repopulation of host-thymus with donor-derived cells when assessed at 4 wk after reconstitution. Lymphocytes from the regenerating thymus of these animals were shown to contain high levels of CTLp which were donor-derived. On the other hand, when the recipient mice were reconstituted with bone marrow cells from donor mice which had been split-dose irradiated 1 mo earlier, regeneration of the recipient thymus was severely depressed when assessed at 4 wk to 3 mo after reconstitution. Although variable but small numbers of donor-derived Thy-1+ cells were detected, CTL activity for alloantigen could not be induced in these donor-derived cells. The results suggest that T cell precursors derived from split-dose irradiated donor mice were unable to undergo active proliferation and differentiation into functional CTLp. The significance of these findings on radiation-induced thymic leukemogenesis is discussed.     

Separation of spleen colony-forming units and prothymocytes by use of a monoclonal antibody detecting an H-2K determinant

The density of H-2K antigens was determined on both the mouse hemopoietic stem cell, using an assay for spleen colony-forming units (CFU-S), and the prothymocyte, using a thymus repopulation assay. This was done by light-activated cell sorting of bone marrow cells labeled first with a biotinylated antibody against H-2Kk and then with avidin-fluorescein isothiocyanate. Almost all CFU-S were found to be present among the 4% bone marrow cells with high forward light scatter (FLS), low perpendicular light scatter (PLS), and bright immunofluorescence. Thymus regeneration by this brightly fluorescent fraction was delayed 3 days compared to thymus regeneration by unsorted cells, although the same number of CFU-S was present in each cell suspension. This delay indicates that differentiation from CFU-S to prothymocytes takes 3 days. The fraction of cells in the FLS/PLS window with dull anti-H-2Kk fluorescence contained few CFU-S and gave rise to a transient thymus regeneration. These findings indicate that the prothymocyte carries fewer H-2K antigens than does the CFU-S. The H-2K antigen is a marker with which CFU-S and prothymocytes can be separated. Therefore, during early T-cell differentiation, the number of H-2K molecules on the cell surface decreases (CFU-S----prothymocyte----cortical thymocyte). During maturation of T cells, a reexpression of H-2K molecules occurs, since lymph node cells and spleen cells were shown to be brightly positive for H-2K antigen.