Molecular pathways in bone marrow homing: dominant role of alpha(4)beta(1) over beta(2)-integrins and selectins.

The specific retention of intravenously administered hemopoietic cells within bone marrow is a complex multistep process. Despite recent insights, the molecular mechanics governing this process remain largely undefined. This study explored the influence of beta(2)-integrins on the homing to bone marrow and repopulation kinetics of progenitor cells. Both antifunctional antibodies and genetically deficient cells were used. In addition, triple selectin-deficient mice were used as recipients of either deficient (selectin or beta(2)) or normal cells in homing experiments. The homing patterns of either beta(2) null or selectin null cells into normal or selectin-deficient recipients were similar to those of normal cells given to normal recipients. Furthermore, spleen colony-forming units and the early bone marrow repopulating activity for the first 2 weeks after transplantation were not significantly different from those of control cells. These data are in contrast to the importance of beta(2)-integrin and selectins in the adhesion/migration cascade of mature leukocytes. The special bone marrow flow hemodynamics may account for these differences. Although early deaths after transplantation can be seen in recipients deficient in CD18 and selectin, these are attributed to septic complications rather than homing defects. However, when beta(2)- or selectin-null donor cells are treated with anti-alpha(4) antibodies before their transplantation to normal or selectin-deficient recipients, a dramatic inhibition of homing (>90%) was found. The data suggest that the alpha(4)beta(1)/vascular cell adhesion molecule-1 pathway alone is capable of providing effective capture of cells within the bone marrow, but if its function is compromised, the synergistic contribution of other pathways, that is, beta(2)-integrins or selectins, is uncovered.

Synergistic mobilization of hemopoietic progenitor cells using concurrent beta1 and beta2 integrin blockade or beta2-deficient mice.

The hierarchy of cytoadhesion molecules involved in hematopoietic/stem progenitor cell mobilization has not yet been delineated. Previous studies have suggested an important role for alpha4beta1 integrin in this process. To test whether mobilization involves dynamic interactions of alpha4beta1 with other integrins on hematopoietic cells, especially the beta2 integrins, mice and primates were treated with anti-beta1 or anti-beta2 antibodies alone or in combination. A single injection of anti-alpha4beta1 antibody elicited reproducible mobilization in contrast to other antibodies, and 3 injections yielded higher mobilization efficiency than each of the other antibodies. When the anti-beta2 (anti-CD11a or anti-CD18) or anti-alpha5/beta1 integrin antibody was combined with anti-alpha4, an augmentation in mobilization was seen that was either additive or synergistic, depending on the potency of the antibody used. Synergy between anti-alpha4 and anti-CD18 (beta(2)) antibody blockade was seen in primates and confirmed in anti-alpha4-treated CD18-deficient mice. In the latter, there was a 49-fold increase in mobilization with anti-alpha4, much higher than in littermate control animals, in CD18 hypomorphic mice, or in other strains of mice tested. Data from both the antibody blockade and gene-targeted mice suggest that the cooperativity of alpha4beta1 with beta2 integrins becomes evident when they are concurrently inhibited. It is unclear whether this cooperativity is exerted at the stage of reversible adhesion versus migration, and enhancement of and whether the 2 integrins work in a sequential or parallel manner. Whatever the mechanism, the data provide a novel example of beta1 and beta2 integrin crosstalk in stem/progenitor cell mobilization.

Mobilization of stem/progenitor cells by sulfated polysaccharides does not require selectin presence.

Employing carbohydrate ligands, which have been extensively used to block selectin function in vitro and in vivo, we have examined the involvement of such ligands in stem/progenitor cell mobilization in mice and monkeys. We found that sulfated fucans, branched and linear, are capable of increasing mature white cells in the periphery and mobilizing stem/progenitor cells of all classes (up to 32-fold) within a few hours posttreatment in a dose-dependent manner. To elicit the effect, the presence of sulfate groups was necessary, yet not sufficient, as certain sulfated hexosamines tested (chondroitin sulfates A or B) were ineffective. Significant mobilization of stem/progenitor cells and leukocytosis was elicited in selectin-deficient mice (L(-/-), PE(-/-), or LPE(-/-)) similar to that of wild-type controls, suggesting that the mode of action of sulfated fucans is not through blockade of known selectins. Other mechanisms have been entertained, in particular, the release of chemokines/cytokines, including some previously implicated in mobilization. Significant increases were documented in the levels of seven circulating chemokines/cytokines within a few hours after fucan sulfate treatment and support such a proposition. Additionally, an increase was noted in plasma metalloproteinase (MMP) 9, which might independently contribute to the mobilization process by enzymatically facilitating chemokine/cytokine release. Mobilization by sulfated polysaccharides provides a distinct paradigm in the mobilization process and uncovers an additional novel in vivo biological role for sulfated glycans. As similarly sulfated compounds were ineffective in vivo, the data also underscore the fact that polysaccharides with similar structures may elicit diverse in vivo effects.

Hemopoietic lineage commitment decisions: in vivo evidence from a transgenic mouse model harboring micro LCR-betapro-LacZ as a transgene.

A substantial body of published data suggests activation of lineage-specific genes in multipotential hemopoietic cells before their unilineage commitment. Because the behavior and plasticity of cells isolated in vitro away from microenvironmental constraints exercised in vivo may be altered, one wonders whether similar findings can be observed in a physiologic setting in vivo. We used a transgenic mouse model harboring human micro LCR together with beta promoter sequences as a transgene to examine activation of lineage-specific programs in vivo. By using LacZ as a reporter, we had the ability to detect, quantitate, and select live cells with different levels of LacZ activation. We found strong expression of LacZ by X-gal staining in 2 lineages-erythroid and megakaryocytic. Activation in the latter was a novel finding not previously observed when similar transgenes were used. We also found activation of muLCR-betapro at low levels in progenitor cells of granulocytic-macrophagic, erythroid, or megakaryocytic lineage detected by in vitro assays, suggesting activation before commitment to a specific lineage pathway. In particular, the expression of LacZ was graded among progenitors, so that in a proportion of them activation occurred only after commitment to erythroid or megakaryocytic lineage. In addition, we found quantitative reduction in LacZ expression between fetal liver and bone marrow-derived cells, the basis of which is unclear. Collectively our data provide in vivo evidence supporting the view that lineage-specific genes are expressed in a graded fashion in pluripotential cells before their irreversible unilineage commitment. (Blood. 2000;95:1274-1282)

Anti-VLA4/VCAM-1-induced mobilization requires cooperative signaling through the kit/mkit ligand pathway.

Although a large body of data on mobilization have yielded valuable clues, the mechanism(s) dictating egress of stem/progenitor cells during baseline hematopoiesis and after their mobilization are poorly understood. We have previously provided functional in vivo evidence that cytoadhesion molecules, specifically the beta1 integrins, are involved in mobilization; however, the mechanism by which this was achieved was unclear. To provide further insights into the anti-very late antigen 4 (VLA4)/anti-vascular cell adhesion molecule 1 (VCAM-1)-induced mobilization, we used these antibodies to treat mutant mice with compromised growth factor receptor function. We found that mobilization by anti-VLA4 does not depend on a functional granulocyte colony-stimulating factor, interleukin-7 (IL-7), or IL-3alpha receptor. By contrast, the functional kit receptor is required, because W/Wv mice responded minimally, whereas Steel-Dickie (Sl/Sld) responded normally. Both Wv and Sl/Sld mice did not respond to anti-VCAM-1 treatment, in contrast to their +/+ littermates and despite normal levels of VCAM-1 expression in bone marrow cells. The defective response to anti-VCAM-1 in W/Wv mice was corrected after their transplantation with +/+ cells. mev/mev mice showed increased numbers of circulating progenitors before treatment and a heightened response after anti-VLA4 or anti-VCAM-1 treatment. Downmodulation of kit expression was detected in normal bone marrow cells after anti-VLA4 treatment. On the strength of the above findings we conclude that (1) anti-VLA4/VCAM-1-induced mobilization likely requires signaling for stimulation of cell migration; (2) this cooperative signaling involves the kit/kit ligand pathway, and provides a novel example of integrin/cytokine crosstalk; and (3) migration mediated through the kit/kit ligand pathway may be a common contributor to different mobilization stimuli. Dissection of the exact molecular pathways that lead to mobilization remains a future challenge.

Antibodies to VLA4 integrin mobilize long-term repopulating cells and augment cytokine-induced mobilization in primates and mice.

Although the use of cytokine-mobilized peripheral blood stem cells has gained a significant momentum in clinical transplantation, the mobilization schemes practiced are guided by a great deal of empiricism. The mechanism(s) by which cytokines or chemokines, alone or in combination, bring about redistribution of stem/progenitor cells from bone marrow to peripheral blood are poorly understood. Likewise the fate of mobilized stem/progenitor cells and their biological properties are incompletely defined. One of the leading hypotheses to explain the mechanism of cytokine-induced mobilization encompasses the view that cytokines disrupt, directly or indirectly, cytoadhesive interactions of stem/progenitor cells with their bone marrow stroma. Compatible with this view are changes in the expression and/or function of several cytoadhesion molecules, especially integrins, postmobilization, and extensive in vitro experimentation supporting the concept of cytokine/integrin interactions. To provide a further insight on the cytokine/integrin interplay in vivo, we have combined cytokine treatments with anti-integrin treatments for mobilization in primates and mice. We found that anti-VLA4 treatment combined with either granulocyte colony-stimulating factor (G-CSF ) treatment or kit ligand treatment leads to significant enhancement of mobilization efficiency (fivefold to eightfold) well above the levels produced by either cytokine alone or anti-VLA4 treatment alone. Similar enhancement was seen when combinations of cytokines, ie, G-CSF plus kit ligand or G-CSF plus Flt3-ligand were used with anti-VLA4 in primates and mice. Furthermore, when anti-VLA4 was given in 5-Fluorouracil-treated primates, significant numbers of progenitor cells were circulating for several days during the recovery period only in the anti-VLA4 treated animals. These data suggest that (1) the effect of anti-VLA4 on mobilization, when used alone, is unlikely to be mediated by secondary cytokine elaboration in vivo; (2) three different cytokines and their combinations do not appear to influence the in vivo responsiveness to anti-VLA4 in coadministration schemes; (3) even if cytokine treatments on their own exert downmodulation of VLA4 function, the target progenitor cells influenced by anti-VLA4 or by cytokines may not necessarily overlap; and (4) augmentation of mobilization in cytokine/anti-VLA4 treatments is most likely caused by an amplification of the pool of target cells on which anti-VLA4 exerts its effects. Because cytokines or anti-VLA4 are each capable of mobilizing long-term repopulating cells and because we show with the present studies that anti-VLA4 in an autologous bone marrow cell transplantation setting does not cause any delay in engraftment, the combination of cytokine/anti-integrin treatment enhancing mobilization may have a clinical use.

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.

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.

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.

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.

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.

Cycling patterns of hemopoietic stem cell subpopulations in young and old BDF1 mice.

We have previously reported that two or more different subpopulations of bone marrow stem cells exist in mice as determined by cycling status of day-8 and day-14 CFU-S in long term bromodeoxyuridine (BrdU) infusion studies. In the present report, comparisons between cycling of stem cell subpopulations in old and young mice show that, while the general patterns persist, there are some statistically significant differences between corresponding time points of early and late CFU-S cycling patterns in young and old BDF1 mice. In both populations of CFU-S there exist cells which do not enter cycle over a five week period. The method which we have employed allowed the cycling measurements to be made in unstimulated steady-state bone marrow cell populations, since no cell death is caused in vivo.

Evaluation of stem cell reserve using serial bone marrow transplantation and competitive repopulation in a murine model of chronic hemolytic anemia.

Serial transplantation and competitive repopulation were used to evaluate any loss of self-replicative capacity of bone marrow stem cells in a mouse model with increased and persistent hemopoietic demands. Congenic marrows from old control and from young and old mice with hereditary spherocytic anemia (sphha/sphha) were serially transplanted at 35-day intervals into normal irradiated recipients. Old anemic marrow failed or reverted to recipient karyotype at a mean of 3.5 transplants, and young anemic marrow reverted at a mean of 4.0 transplants, whereas controls did so at a mean of 5.0 transplants. In a competitive assay in which a mixture of anemic and control marrow was transplanted, the anemic marrow persisted to 10 months following transplantation; anemic marrow repopulation was greater if anemic marrow sex matched with the host. It is possible that lifelong stress of severe anemia decreases stem cell reserve in the anemic sphha/sphha mouse marrow. However, marginal differences in serial transplantation number and the maintenance of anemic marrow in a competition assay would suggest that marrow stem cells, under prolonged stress, are capable of exhibiting good repopulating and self-replicating abilities.

Kinetics of early and late spleen colony development.

The kinetics of spleen colony development were studied with special emphasis upon the self-replicative capacity of CFU-S, which are responsible for the early- or late-appearing spleen colonies, and upon CFU-S migration in the days following transplantation. It was found that, while the spleen colonies had a much lower daughter CFU-S content at eight days than at 11 or 12 days, many of the 8-day colonies remained in the spleen to become 12-day colonies which had many daughter CFU-S. In addition, it was found that large numbers of CFU-S were present in the splenic space between the colonies. Evidence was developed that these "intercolonial space" CFU-S were the founders of many of the late-appearing spleen colonies and that many or all of them were transported from the bone marrow to the spleen via the blood at varying times after the transplantation. Approximately one-half of the day-12 colony founders were found to arrive in the spleen after the third posttransplantation day.

Normal cycling patterns of hematopoietic stem cell subpopulations: an assay using long-term in vivo BrdU infusion.

It was found in a long-term bromodeoxyuridine (BrdU) infusion study that two or more different subpopulations of bone marrow stem cells exist in mice. One of these subpopulations appears to be noncycling and forms approximately 10% of eight-day CFU-S. Another one, a subpopulation of slowly cycling bone marrow cells, is represented as 14-day CFU-S. The 14-day CFU-S have a regular increment in the percentage of the subpopulation entering the cycle over time, with a cell generation half-time of 21 days. The cycling status in these experiments was ascertained by in vivo continuous long-term BrdU infusion. An improved method is presented for long-term BrdU infusion with UV killing of cycled cells.

A technique for the daily examination of spleen colonies in mice.

We have developed a surgical method and an insertable device for viewing the daily changes in number, shape, and development of hemopoietic spleen colonies in mice. With it we have been able to follow the spleen colony changes in individual animals over the period when macroscopic or moderately magnified counts of spleen colonies are customarily made. We have found that about half of the spleen colonies present on day 8 after transplantation do remain through day 12, while an approximately equal number disappear during this period. Further, the colonies that disappear are replaced by an equal or larger number of newly developing colonies at roughly the same temporal sequence as their disappearance. We speculate that these late-appearing colonies may be late arrivals from the recipient's previously seeded bone marrow.

Depletion of reserve in the hemopoietic system: III. Factors affecting the serial transplantation of bone marrow.

The effects of serial transplantation on murine bone marrow were studied. Decline of CFUs content and the maximum number of successful transplantations possible were the criteria considered to indicate stem cell changes accompanying the serial transplants. Variables of experimentation included age of the marrow cell donors, addition of thymocytes to the transplanted marrow, differing time intervals between transplantations and differing numbers of donor marrow cells injected throughout a given transplant series. All of the transplantation series were monitored by karyotyping the marrow cells after each transplantation to document the continuance of the original donor cells. Reversions to recipient cell type did occur late in some series and were considered the end point of these series, as were the disappearance of CFUs and deaths of recipients due to transplant failure (the latter two always corresponded well). Only the donor marrow cell dose was found to be an important variable in the continuance of a transplant series and it was possible to obtain 8 successful serial transplantations with the highest marrow cell dose. All of the series given lower cell doses failed at the fifth or sixth transplant, regardless of other variables involved. A naturally occurring hierarchy of hemopoietic stem cells coupled with stem cell differentiative changes caused by extreme and repeated demands for population replenishment is offered as a probable reason for the results obtained, while other possibilities are discussed as well.

Decline of colony-forming units with serial bone marrow cell passages: intrinsic or extrinsic causation?

A series of experiments were conducted to approach the question of whether CFU-S decline in self-replicative potential and in life saving potential in the serial transplant recipient were the result of events intrinsic to the CFU-S lineage or were the direct result of incidental external events. Specifically, the possibilities of CFU-S death or loss of potential related to in vitro manipulation, related to failure of the most capable CFU-S to lodge in the bone marrow, related to failure of accessory cells to lodge or related to and coincident with increased CFU-S cycling were all examined. Only the last named factor was associated with CFU-S decline. The results were compatible with the generation age concept i.e., that the loss of CFU-S self replicative potential was due to a permanent shift of the most "primitive" CFU-S to a more differentiated and less self-replicative status when an extreme and prolonged demand for replenishment of the more differentiated CFU-S pools exists.