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N S Wolf

Publications and source records attributed to N S Wolf.

At least 19 recordsLinked to original sources
PubMed1997

Long-term caloric restriction delays age-related decline in proliferation capacity of murine lens epithelial cells in vitro and in vivo.

PURPOSE: The goal of this study was to examine the effects of age and long-term caloric restriction on the proliferation capacity of murine lens epithelial (LE) cells in vitro and in vivo. METHODS: B6D2F1 (C57BL/6 X DBA/2) F1 mice 4 to 45 months of age were obtained and fed either an ad libitum (AL) or a calorically restricted (CR) diet (60% of AL intake). Cellular proliferation capacity in vitro was measured using the colony size distribution assay for 10-day clonal growth of mouse LE cells. Proliferation rate in vivo was assayed using immunostaining for 5-bromo-2'-deoxyuridine (BrdU) in mouse LE cells after 2-week osmotic pump delivery of BrdU. RESULTS: Proliferative capacity of cells from old AL mice decreased significantly in comparison to cells from young AL and old CR mice, as determined by the fractions of cells capable of forming small (no or one cell division) and large (four or more cell divisions) colonies in vitro. There was also a decline in cell replicative rate as measured by BrdU labeling index (LI) in vivo with increasing age in AL and CR mice. However, this decline was marked in AL mice between 10 and 30 months of age and minimal in CR mice. Significant differences in BudU LI between AL and CR mice occurred when animals were 30 months of age or older. This finding indicates that an age-related decline in cellular proliferation rate in vivo was delayed by CR. CONCLUSIONS: A significantly reduced proliferative capacity of LE cells is associated with increased age of mice and is delayed by long-term caloric restriction as measured in vitro and in vivo. How caloric restriction mediates its effects on LE cell proliferation remains to be investigated further.

Aging↗
PubMed1996

Enhanced cell proliferation and biosynthesis mediate improved wound repair in refed, caloric-restricted mice.

Aged mice that have undergone long-term caloric-restriction (CR) have improved health and enhanced longevity in comparison to aged mice that are ad libitum-fed (AL). However, caloric-restriction does not benefit the impaired wound healing of aged mice. To test the hypothesis that CR mice have the capacity for enhanced wound repair, but require a short-term period of additional nutrient intake to show this advantage, we assessed wound healing in CR mice that had been refed (RF) an ad libitum diet for 4 weeks prior to wounding. Two strains of AL young (Y AL) (4-6 months), AL middle-aged (M AL) (15-17 months), and three different, matched cohorts of old mice (O) (30-33 months): O AL, O CR, and O RF were studied. Two full-thickness 4 mm diameter punch biopsy skin wounds were created on the dorsum of each mouse. Animals were sacrificed and wounds were harvested at 1,2,3,5, and 7 days post-wounding. Repair of wounds was slower in O AL and O CR mice compared to Y AL and M AL animals. In contrast, the O RF mice healed similarly to that of the Y AL and M AL mice, as assessed by measures of wound area and histologic criteria. O RF mice demonstrated enhanced synthesis of type I collagen mRNA in comparison to O AL and O CR mice. A greater number of endothelial cells and fibroblasts at the wound edge of the O RF mice exhibited replication in vivo as measured by uptake of BrdU. O RF mice had higher levels of insulin-like binding protein 3 (IGFBP-3). Furthermore, fibroblasts derived from the explant of the punch biopsy of O CR mouse skin revealed enhanced proliferation and contraction in vitro, in comparison to fibroblasts from the O AL mice. In conclusion, O RF mice demonstrate an enhanced capacity to undergo wound repair in comparison to O AL mice. This effect appears to be mediated, in part, by enhanced cell proliferation, contraction, and collagen biosynthesis. In addition, short-term refeeding induced an increase in the serum level of IGFBP-3, the major binding protein for IGF-1. These data confirm that cells from O CR animals have a preserved proliferative, biosynthetic, and contractile capacity, but that an adequate source of nutrients is necessary to demonstrate this advantage in wound healing.

Aging↗
PubMed1996

Interaction of stem cell factor and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen.

The phenotypes of mice that harbor a defect in the genes encoding either stem cell factor (SCF) or its receptor, c-kit, indicate that this ligand/receptor pair is necessary for maintenance of normal hematopoiesis in the adult. Our objective was to determine whether SCF, like erythropoietin, is necessary for acute erythroid expansion during recovery from hemolytic anemia. Monoclonal antibody ACK2, which recognizes the murine c-kit receptor, was used to selectively block the hematopoietic growth-promoting effects of SCF. Mice were treated with phenylhydrazine on day 0 and day 1 to induce hemolytic anemia and also received no antibody, control IgG, or ACK2 on day 0. The mice were killed on day 3 and the hematocrit (Hct), reticulocyte count, and numbers of erythroid and myeloid hematopoietic progenitor cells (colony-forming unit-erythroid [CFU-E], burst-forming unit [BFU]-E, and CFU-granulocyte-macrophage [GM]) were quantitated in the femoral marrow and spleen using hematopoietic colony-forming assays. Induction of hemolytic anemia with phenylhydrazine resulted in a drop in the Hct from approximately 50% to 30%, and an approximate 8- to 10-fold increase in the reticulocyte count. The numbers of CFU-E increased modestly in the femur, and approximately 25- to 50-fold in the spleen, in comparison with normal mice. BFU-E and CFU-GM values did not increase in the femur but expanded 6- to 10-fold in the spleen, in comparison with normal mice. This confirms that much of the erythroid expansion in response to hemolytic anemia occurs in the murine spleen. Neutralizing quantities of the ACK2 antibody reduced femoral CFU-E, BFU-E, and CFU-GM content to less than half that found in phenylhydrazine-treated control mice and nearly totally ablated splenic hematopoiesis. These results suggest that c-kit receptor function may be required for optimal response to acute erythropoietic demand and that erythropoiesis in the splenic microenvironment is more dependent on SCF/c-kit receptor interaction than is erythropoiesis in the marrow microenvironment. Because expansion of late erythropoiesis in the spleen was preferentially blocked, we tested the hypothesis that homing of more primitive hematopoietic cells to the spleen was dependent on c-kit receptor function. Lethally irradiated mice were injected with marrow cells obtained from mice that had received phenylhydrazine plus control IgG or with marrow cells obtained from mice that had received phenylhydrazine plus ACK2. In parallel experiments, normal murine marrow cells were treated in vitro with control IgG or with ACK2 and were injected into lethally irradiated mice. The fraction of BFU-E and CFU-GM retrieved from the marrow and spleen of the recipient mice 4 hours later was reduced by approximately 75% when progenitor cells had been exposed to ACK2, in comparison with control IgG. These data suggest that interaction of SCF with the c-kit receptor affects the homing behavior of hematopoietic progenitor cells in the adult animal.

Anemia, Hemolytic↗
PubMed1996

Transforming growth factor beta 1 directly and reversibly inhibits the initial cell divisions of long-term repopulating hematopoietic stem cells.

Hematopoiesis appears to be regulated, in part, by a balance between extracellular positive and negative growth signals. Transforming growth factor beta-1 (TGF-beta 1) has been shown to be a negative regulator of primitive hematopoietic cells. This study examined the direct effect of TGF-beta 1 on the proliferation and differentiation of long-term repopulating hematopoietic stem cells (LTR-HSC) in vitro. We previously reported a cell fractionation approach that includes the selection of low Hoescht 33342/low Rhodamine 123 (low Ho/Rh) cell fractions that are highly enriched for long-term repopulating cells (LTR-HSC) and also clone to a very high efficiency in the presence of stem cell factor (SCF) + interleukin-3 (IL-3) + IL-6: 90% to 100% of individually cultured low Ho/Rh cells formed high proliferative potential clones. This high cloning efficiency of an LTR-HSC enriched cell population enabled proliferation inhibition studies to be more easily interpreted. In this report, we show that the continuous presence of TGF-beta 1 directly inhibits the cell division of essentially all low Ho/Rh cells (in a dose-dependent manner) during their 0 to 5th cell division in vitro. Therefore, it follows that TGF-beta 1 must directly inhibit the proliferation of LTR-HSC contained within these low Ho/Rh cells. The time required for some low Ho/Rh cells to undergo their first cell division in vitro was also prolonged in the presence of TGF-beta 1. Furthermore, when low Ho/Rh cells were exposed to TFG-beta 1 for varying lengths of time before neutralization of the TGF-beta 1 by monoclonal antibody, the ability to form macroclones was markedly decreased after approximately 4 days of TGF-beta 1 exposure. In addition, 1 to 10 ng/mL of TGF-beta 1 resulted in a maintenance of high proliferative potential-colony-forming cell (HPP-CFC) during 8 days of culture compared with loss of HPP-CFC in cultures with no added TGF-beta 1. In conclusion, this study shows that TGF-beta 1 directly inhibits the initial stages of proliferation of LTR-HSC and appears to slow the differentiation of daughter cells of low Ho/Rh cells.

Animals↗
PubMed1996

The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells.

In this study, we explored whether thrombopoietin (Tpo) has a direct in vitro effect on the proliferation and differentiation of long-term repopulating hematopoietic stem cells (LTR-HSC). We previously reported a cell separation method that uses the fluorescence-activated cell sorter selection of low Hoescht 33342/low Rhodamine 123 (low Ho/low Rh) fluorescence cell fractions that are highly enriched for LTR-HSC and can reconstitute lethally irradiated recipients with fewer than 20 cells. Low Ho/low Rh cells clone with high proliferative potential in vitro in the presence of stem cell factor (SCF) + interleukin-3 (IL-3) + IL-6 (90% to 100% HPP-CFC). Tpo alone did not induce proliferation of these low Ho/low Rh cells. However, in combination with SCF or IL-3, Tpo had several synergistic effects on cell proliferation. When Tpo was added to single growth factors (either SCF or IL-3 or the combination of both), the time required for the first cell division of low Ho/low Rh cells was significantly shortened and their cloning efficiency increased substantially. Moreover, the subsequent clonal expansion at the early time points of culture was significantly augmented by Tpo. Low Ho/low Rh cells, when assayed in agar directly after sorting, did not form megakaryocyte colonies in any growth condition tested. Several days of culture in the presence of multiple cytokines were required to obtain colony-forming units-megakaryocyte (CFU-Mk). In contrast, more differentiated, low Ho/high Rh cells, previously shown to contain short-term repopulating hematopoietic stem cells (STR-HSC), were able to form megakaryocyte colonies in agar when cultured in Tpo alone directly after sorting. These data establish that Tpo acts directly on primitive hematopoietic stem cells selected using the Ho/Rh method, but this effect is dependent on the presence of pluripotent cytokines. These cells subsequently differentiate into CFU-Mk, which are capable of responding to Tpo alone. Together with the results of previous reports of its effects on erythroid progenitors, these results suggest that the effects of Tpo on hematopoiesis are greater than initially anticipated.

Animals↗
PubMed1996

Aged murine T-lymphocytes are more resistant to oxidative damage due to the predominance of the cells possessing the memory phenotype.

Glutathione (GSH) is the most important cytosolic antioxidant. Since GSH levels are decreased with age, we hypothesized that T-lymphocytes from old mice would be more sensitive to oxidative stress. T-lymphocytes from young and old mice were exposed to hypoxanthine/xanthine oxidase, and lymphocyte viability, proliferation, GSH content, and calcium signaling were measured. Before exposure, proliferation of T-lymphocytes from young mice was greater than that of old; following exposure, the converse was true. This was in spite of the fact that old mice had lower total GSH levels and greater levels of glutathione disulfide. After oxidative challenge, intracellular calcium responses to anti-CD3 were decreased in naive T-lymphocytes from all mice, while memory lymphocytes were less affected. Higher proportions of memory lymphocytes in old mice resulted in their greater overall preservation of lymphocyte function following oxidative injury, contrary to expectations that lower lymphocyte GSH content with age would increase susceptibility to oxidative stress.

Aging↗
PubMed1995

The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen.

Selective lodgement or homing of transplanted hemopoietic stem cells in the recipient's bone marrow (BM) is a critical step in the establishment of long-term hemopoiesis after BM transplantation. However, despite its biologic and clinical significance, little is understood about the process of homing. In the present study, we have concentrated on the initial stages of homing and explored the functional role in vivo of some of the adhesion pathways previously found to mediate in vitro adhesion of hemopoietic cells to cultured BM stroma. We have found that homing of murine hemopoietic progenitors of the BM of lethally irradiated recipients at 3 h after transplant was significantly reduced after pretreatment of the donor cells with an antibody to the integrin very late antigen 4 (VLA4). This inhibition of marrow homing was accompanied by an increase in hemopoietic progenitors circulating in the blood and an increased uptake of these progenitors by the spleen. Similar results were obtained by treatment of the recipients with an antibody to vascular cell adhesion molecule 1 (VCAM-1), a ligand for VLA4. Furthermore, we showed that administration of the same antibodies (anti-VLA4 or anti-VCAM-1) to normal animals causes mobilization of hemopoietic progenitors into blood. These data suggest that hemopoietic cell lodgement in the BM is a regulatable process and can be influenced by VLA4/VCAM-1 adhesion pathway. Although additional molecular pathways are not excluded and may be likely, our data establish VCAM-1 as a BM endothelial addressin, analogous to the role that mucosal addressin cell adhesion molecule (MAdCAM) plays in lymphocyte homing. Whether splenic uptake of hemopoietic progenitors is passive or controlled through different mechanisms remains to be clarified. In addition, we provide experimental evidence that homing and mobilization are related phenomena involving, at least partly, similar molecular pathways.

Animals↗
PubMed1995

Influence of age, sex, and dietary restriction on intracellular free calcium responses of CD4+ lymphocytes in rhesus monkeys (Macaca mulatta).

The influence of aging and dietary restriction on increase in intracellular free calcium ([Ca2+]i) of CD4+ lymphocytes from Macaca mulatta was examined after stimulation with anti-CD3 mAb. We used a flow cytometric assay with the dye indo-1 and either direct or reciprocal immunofluorescent staining to identify CD4+ cells. After stimulation with anti-CD3 mAb, intracellular free calcium responses were reduced in CD4+ lymphocytes from old male and female ad libitum fed monkeys compared to young and adult male or female monkeys. Old female monkeys had significantly lower [Ca2+]i than did old male monkeys. The reduced responses were in part related to a decreased percentage of responding cells. Dietary restriction of males over a four-year period did not alter [Ca2+]i response compared to ad libitum fed male monkeys. Female monkeys of all ages (which were restricted only for four months) also had similar [Ca2+]i responses to ad libitum fed controls. Our data suggest that age-related changes in [Ca2+]i responses are similar between humans and M. mulatta, and that over these intervals, no effects of caloric restrictions can be detected.

Aging↗
PubMed1995

Support versus inhibition of hematopoiesis by two characterized stromal cell types.

Stromal cells are believed to regulate hematopoiesis through direct cell-cell contact interactions and the release of growth factors. Many questions remain, however, about their lineage derivation and functional heterogeneity. We have previously shown that the adherent nontransformed, nonimmortalized murine bone marrow stromal cell population consists of three cell types which could be grown separately in vitro. Based on the phenotype characterization and expression of surface antigens, we proposed a classification listing for murine bone marrow stromal cells as macrophages, endothelial-like cells and myofibroblasts that display smooth muscle-like characteristics in culture. The present study describes the ability of each of these freshly isolated separated murine stromal cell populations to support the growth of primitive hematopoietic stem cells previously characterized as highly enriched in long-term repopulating cells (LTRC). Of the three stromal cell types tested only the myofibroblasts were capable of support for multilineage hematopoiesis derived in vitro from LTRC in a cloning ring culture system. Endothelial-like cells had an inhibitory effect on the proliferation of LTRC and their descendant cells that was induced by exogenous growth factors. This inhibitory activity was present in a low molecular weight filtrate of endothelial-like cells culture medium. This suggests an essential role for marrow stroma myofibroblasts in the support of proliferation of hematopoietic cells at the stage of early divisions of primitive hematopoietic stem cells and endothelial-like cells as negative regulators of this proliferation.

Alkaline Phosphatase↗
PubMed1995

Caloric restriction: conservation of cellular replicative capacity in vitro accompanies life-span extension in mice.

We have tested whether life-long caloric restriction (CR) slows or delays the age-related loss of cellular replicative potential that occurs during normal aging in ad libitum (AL) fed mice. Both mean and maximum life spans of the restricted animals (60% of AL intake) were significantly extended 30-40% by CR treatment. Proliferative potential, measured by determining the fraction of cells capable of forming large clones in vitro, was compared in five cell types from six tissue sites from two strains of mice (Male (C57BL/6 x DBA/2)F1("B6D2F1") and female (C57BL/6 x C3H)F1("B6C3F1")). This included four nonhematopoietic organ sites: fibroblast cells from ear skin, tail skin, and subdermal connective tissue and epithelial cells from the medullary part of the kidney and two cell types, myofibroblasts and endothelial-like cells, from spleen and bone marrow. The proliferative potential of cells from AL mice decreased progressively with age in all tissues sites of both mouse strains. CR delayed or decreased the loss of proliferative potential in all situations, but the timing of this was tissue specific. For cells from the four nonhematopoietic tissues sites from female B6C3F1 female mice, CR delayed the onset of proliferative loss, such that the fraction of large clones was significantly greater for the CR 18- to 24-month-old mice than in AL controls at three of four sites (as determined by the fraction of large clones after 1 week of clonal growth). The proliferative loss in CR tissues then accelerated from 24 to 30 months, so that both CR and AL mice had similar fractions of large clones after 30 months of age. CR was also seen to delay loss of proliferative potential in cells from skin and kidney of B6D2F1 male mice at 23-24 months of age when cloned for 2 weeks. For fibroblast and endothelial-like cells from bone marrow and spleen stromal sites from both strains of mice, CR also significantly decreased loss of proliferative potential; furthermore, in these tissues the proliferative advantages remained or increased from 24 to over 30 months of age. In companion studies (N.S. Wolf et al., 1995. Exp. Cell. Res. 217, 000-000), CR was seen to decrease age-related losses in the maximal rates of cell replication in vivo in a panel of tissues from B6D2F1 male mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Caloric restriction: conservation of in vivo cellular replicative capacity accompanies life-span extension in mice.

In male mice of a long-lived hybrid strain (B6D2F1), long-term 40% caloric restriction (CR) extended both mean and maximum life spans by 36 and 20%, respectively, over that of ad libitum fed (AL) controls. Measurements of entry into S-phase were made in vivo of six different cell types in five different organs using 2-week exposures to BrdU. The labeling index (L.I.) in all organs studied was lower in young CR mice than in young AL fed mice. In most cases, the L.I. in AL mice fell to the levels of that in the CR mice by 13 months of age, and the two groups then remained so through old age. However, when the L.I. was measured in old CR mice which had been placed on the AL diet for a period of 4 weeks (this was termed refeeding (RF), it was found to be above that of similar age AL or CR mice and almost at the level of young AL mice. This was still true, but to a lesser degree, in a repeat study using an 8-week period of RF. In a separate but parallel in vitro study (companion paper, this volume), the superiority of CR over AL for retention of cellular replication capacity was confirmed by clone size distribution measurements made in several cell types in mice of several age groups. These results indicate that: (1) the rate of cell replication in AL diet mice diminishes greatly by early middle age in all organ sites studied and then plateaus or declines much more slowly; (2) CR broadly preserves in vivo cellular replicative capacity but often requires the energy levels provided by a switch to AL feeding to demonstrate this late in life; (3) accordingly, the replicative deficit in AL fed mice appears to be cumulative and is significant only in old age. The mechanism(s) involved is yet to be discovered but may be related to, or even the same as, that which extends life spans in CR animals. Correspondingly, and with corroborative data from our in vitro companion study, (W. R. Pendergrass et al., 1995. Exp. Cell. Res. 217, 309-316), we suggest that cell populations sustain an accrual of biochemical damage or physiological alterations which increasingly limit their replicative capacity as the animal ages, and that CR reduces the accrual of this damage.

Aging↗
PubMed1995

Developmental hematopoiesis from prenatal to young-adult life in the mouse model.

Five measurements of hematopoietic function were made in the mouse from midfetal life to young adulthood. These included two in vivo (day-8 colony-forming unit-spleen [CFU-S8] and day-12 CFU-S [CFU-S12]) and two in vitro clonal measurements of hematopoietic stem and progenitor cells (high proliferative potential colony-forming cell [HPP-CFC] and CFC of low proliferative potential [LPP-CFC]) as well as an in vitro clonal measurement of colony-forming unit-fibroblast (CFU-F). The appearance, increase, subsequent decrease, and later emergence and increase of each of these parameters in the fetal-liver, newborn, growing-infant, and young-adult bone marrow were correlated and found to be in parallel. Exceptions to this included the earlier appearance in the fetal liver of CFU-F and the relatively differentiated hematopoietic LPP-CFC. The pattern of emergence of these progenitor cell subpopulations in the fetal liver may be related, in part to the timing of the hematopoietic microenvironment development and the relative frequencies of progenitor cell types in the circulation. This developmental study in the mouse model describes additional correlations between in vivo and in vitro colony-forming stem cells and fibroblastic stromal colony-forming cells, and it suggests the dependence of hematopoietic stem cells upon the stromal microenvironment for the necessary conditions for hematopoietic stem cell lodgment, growth, and maturation.

Aging↗
PubMed1993

Dissecting the hematopoietic microenvironment. IX. Further characterization of murine bone marrow stromal cells.

We have previously shown the adherent nontransformed, nonimmortalized murine bone marrow stromal cell (BMSC) population to consist of phagocytic macrophage and endothelial-like cells and nonphagocytic fibroblasts. Both colonial and near confluent growth of each cell type was obtained following magnetic bead separation, subsequent passaging, and sustained culture with fetal bovine serum and cytokines. Monoclonal antibody staining of antigenic determinants was used to characterize the phenotype of the stromal cell population in primary platings of murine colony-forming unit fibroblast and long-term bone marrow cultures. The antibodies MECA-99, MECA-32, and MJ7-18, raised against murine vascular endothelial antigenic determinants, and von Willebrand's factor all stained selectively for the rounded endothelial-like cells. Endothelial-like cells as well as macrophages expressed the myeloid surface antigens F4/80, 7/4, and Mac-1 under our culture conditions. The cytoskeleton of the stromal fibroblasts in culture was shown to express smooth muscle-specific actin isoforms, as evidenced by positive staining of stress fibers for alpha smooth muscle-1, CGA-7 (alpha/gamma isoforms), and HHF-35 (recognizes all muscle-specific actins). Under culture conditions, stromal fibroblasts were also found to be positive for a polyclonal smooth muscle myosin. It was found that these fibroblasts stained for collagens type I, III, and IV in our cultures. Although collagen type IV is considered a by-product of endothelial cells, endothelial-like cells in our cultures did not stain for any of the collagen types. We propose a classification listing for murine BMSCs as macrophages, endothelial-like cells, and fibroblasts that display smooth muscle-like characteristics in culture.

Animals↗
PubMed1993

Decrease in cellular replicative potential in "giant" mice transfected with the bovine growth hormone gene correlates to shortened life span.

Adult mice, (C57BL/6 x Sjl)F1 hybrids, transfected with the bovine growth hormone gene (bGH) grow to twice normal size, but have a mean life span less than 50% that of control siblings without the transgene. The replicative potentials of cells from six different tissue sites (tail skin and ear skin dermal fibroblasts, tail subdermal connective tissue fibroblasts, kidney medulla epithelial cells, bone marrow myofibroblasts, and spleen myofibroblasts) were assayed in vitro using clone size distribution analysis. Cells from all of the above bGH+ tissues produced a smaller fraction of large clones, relative to age-matched controls, in all of these cell types. The loss of replicative potential did not appear to be the result of negative conditioning of the cloning media by the bGH+ cells, and was tightly correlated to the period of accelerated growth in these animals (3-12 weeks), a time when additional GH receptors are expressed.

Age Factors↗
PubMed1993

In vivo and in vitro characterization of long-term repopulating primitive hematopoietic cells isolated by sequential Hoechst 33342-rhodamine 123 FACS selection.

Subpopulations of very primitive hematopoietic cells were isolated by fluorescence-activated cell sorter (FACS) selection of density gradient-enriched, lineage-depleted marrow cells with blast cell light scatter characteristics that bound low levels of the DNA binding dye, Hoechst 33342 (Hö) and retained differential amounts of the mitochondrial binding dye, rhodamine 123 (Rh-123). The dyes were used sequentially in a single sorting operation. The subfractions of cells that stained most weakly with both dyes were highly coenriched for long-term repopulating cells (LTRC) and for in vitro high proliferative potential colony-forming cells (HPP-CFC). Furthermore, as populations of cells were progressively selected on the basis of decreasing Hö and Rh-123 fluorescence, first the CFU-S-8, then the CFU-S-12 diminished or disappeared entirely in the lowest Rh-123 fraction. In these low fluorescent populations, plating efficiency for HPP-CFC was very high when cultured in the combined presence of recombinant rat stem cell factor (rrSCF), recombinant human interleukin-1 (rhIL-1), recombinant murine interleukin-3 (rmIL-3) and recombinant human colony-stimulating factor-1 (rhCSF-1), apparently reaching 100% in some instances. When 20 male donor cells from this lowest fluorescent Hö/Rh-123 fraction were injected into lethally irradiated female recipients, along with a "compromised" marrow cell population (3x previously transplanted nonsorted female bone marrow cells), the sorted male donor cells were able to completely and exclusively repopulate the myeloid and the lymphoid B and T cell compartments of the recipients for at least 10 months posttransplant. Assays of cell fractions that were relatively more Rh-123 fluorescent demonstrated the presence of cell with progressively less repopulating capacity. When descendants of transplanted low fluorescent Rh-123 selected cells, as found in 12-day spleen colonies, were assayed for the capacity to provide long-term survival in secondary recipients, they were able to do so in a high proportion of lethally irradiated recipients. However, spleen colonies derived from the mid-high fluorescence fraction were completely unable to do so. In summary, we have demonstrated with a sequential Hö/Rh-123 sorting system that a subset of HPP-CFC cofractionate with LTRC with high frequency. Using this system, the enrichment of LTRC in the lowest Rh-123 compartment of the sequentially Hö/Rh-123 selected cells appears to be the greatest demonstrated thus far. In addition, this study further supports previous ones that identify a compartment of LTRC that are largely distinct from CFU-S-12.

Animals↗
PubMed1992

An age-related reduction in the replicative capacity of two murine hematopoietic stroma cell types.

Two stromal cell types, myofibroblasts and endothelial-like cells, that were identifiable by structural and antigenic specificities, were obtained from murine bone marrow and spleen of young, middle-aged, and old mice of two strains and sexes and grown in liquid culture for 9 or 10 days. As expected, there were more total nucleated cells per organ in the old mice (with larger organs) than in the young mice. However, the concentration of stromal colony forming cells was greater in the young mice, resulting in the number of colony forming cells per organ not being significantly different in most comparisons. The in vitro replicative capacity of the two stromal cell types from both organs in all age groups was determined by clone size distribution assays. In all instances the number of cell doublings achieved was statistically significantly greater in the stromal cell clones from young mice than those from old mice. The cell doubling capacity of the middle-aged mice fell between that of the young and the old mice and in most instances that difference was also statistically significant. It was concluded that these in vitro findings constituted a biomarker of aging in these tissues and that this was significant in relation to previous in vivo and in vitro work by these authors and by others reporting the inferiority of aged bone marrow and spleen stroma to regenerate and to support hematopoiesis.

Aging↗
PubMed1990

Transplantation of hematopoietic stem cells obtained by a combined dye method fractionation of murine bone marrow.

Hematopoietic stem cells were purified from murine bone marrow cells (BMC). Their characteristic density, size, internal complexity, Hoechst 33342 dye uptake, and wheat germ agglutinin (WGA) affinity were used to distinguish them from other cells in the bone marrow. BMC suspensions were centrifuged over Ficoll Lymphocyte Separation Media (Organon Teknika, Durham, NC; density 1.077 to 1.08). The lower-density cells were drawn off, stained with Hoechst and labeled with biotinylated WGA bound to streptavidin conjugated to phycoerythrin (WGA-B*A-PE) or with WGA conjugated to Texas Red. These cells were then analyzed and sorted by an Ortho Cytofluorograph 50-H cell sorter. The cells exhibiting medium to high forward light scatter, low to medium right angle light scatter, low Hoechst intensity, and high WGA affinity were selected. Sorted BMC (SBMC) were stained with Romanowsky-type stains for morphologic assay, and were assayed in lethally irradiated (LI) mice for their ability to produce colony-forming units in the spleen (CFU-S) and for their ability to produce survival. The spleen seeding factor for day 8 CFU-S upon retransplantation of the isolated cells was 0.1. The isolated cells were found to have consistent morphology, were enriched up to 135-fold as indicated by day 8 CFU-S assay, 195-fold as indicated by day 14 CFU-S assay, and 150 sorter-selected BMC were able to produce long-term survival in LI mice with retention of donor karyotype. When recipients of this first transplantation were themselves used as BMC donors, their number of day 8 and day 12 CFU-S were found to be reduced. However, 3 X 10(5) of their BMC provided 100% survival among secondary recipients. When the previously SBMC were competed after one transplantation against fresh nonsorted BMC in a mixed donor transplant, they showed the decline in hematopoietic potency normally seen in previously transplanted BMC. We conclude that the use of combinations of vital dyes for fluorescence-activated cell sorting (FACS) selection of survival-promoting murine hematopoietic stem cells provides results comparable with those produced by antibody-selected FACS and has the advantage of a method directly transferable to human BMC.

Animals↗