Prolonged Effects of Nitrogen Mustard on Mouse Haematopoietic and Lymphoid Tissues

Groups of mice have been autopsied at regular intervals during the period of lymphomyeloid tissue regeneration which follows the phase of hypocellularity induced by the i.v. injection of 100 μg (4 mg/kg bodyweight) nitrogen mustard. The marrow cellularity recovered to levels in the normal range by the 8th day and remained in this range up to the 40th day. Subsequently, the marrow showed a slight degree of hypocellularity up to day 120. Granulocytes were predominant during the initial phase of marrow regeneration from days 5–12. The thymus, lymph nodes, and spleen commenced regeneration during the second week post-injection. The thymus exhibited periodic size variations such that it was substantially larger than the thymus of age-matched controls from 20–30 d, 46–73 d, and 75–100 d after injection. The lymph nodes and lymphoid tissue of the spleen regenerated only slowly to reach control values by 40–50 d. Superimposed on the recovering lymphoid tissue of the spleen was a phase of erythroid hyperplasia lasting from 10–18 d post-injection. This coincided with a shift from granulocytosis to erythroid hyperplasia in the marrow. This erythroid hyperplasia lasted until day 30 when the cellular composition of the marrow and spleen returned to normal. A possible explanation of these results is that nitrogen mustard introduces a degree of synchrony into stem cell proliferation and differentiation. Additionally, these results emphasize the role of the haematopoietic microenvironment in the control of stem cell differentiation.     

Inverse Relationship between Splenomegaly and Stem Cell Compartment Size in Mice Treated with Nitrogen Mustard

Following the administration of similar doses of nitrogen mustard (4 mg/kg) to different strains of mice, wide variations in the subsequent degree of splenomegaly were observed, implying strain differences in the role of the spleen in the compensatory erythropoietic response to haematopoietic stress. This investigation was undertaken to determine whether or not these differences were related to the size of the haematopoietic stem cell compartment size in the various strains of mice. Groups of 4 different strains of mice (Swiss Webster, A/J, C57BL/6J and CS1/ASH) were injected i.v. with nitrogen mustard (4 mg/kg body weight) and autopsied at regular intervals up to 20 d post-injection. At autopsy, the wet weight of the spleen was determined. Subsequently, groups of the same 4 strains of mice were exposed to single doses of wholebody γ-irradiation in the range of 500–900 rads. 9 d after γ-irradiation the mice were autopsied, their spleens removed, and the number of endogenous spleen colonies determined. The greatest degree of splenomegaly was observed in the C57bl/6J mice. The Swiss Webster mice showed no splenomegaly during the time period studied. There existed a linear inverse relationship between the maximum degree of splenomegaly observed and the dose of wholebody γ-irradiation required to completely eliminate endogenous spleen colonies. This data is in accord with the hypothesis that there exists an inverse relationship between the extent of splenomegaly observed following haematopoietic stress and the haematopoietic stem cell compartment size.     

HEMATOPOIESIS IN PANTOTHENIC ACID-DEFICIENT RATS

Rats fed a purified diet low in pantothenic acid developed granulocytopeniaand anemia singly or in combination. In the former, the marrow showed markeddepletion of granulocytes, particularly of the more mature cells, and a slight increase in erythroid cells. In combined granulocytopenia and anemia the granulocytes of the marrow were still further reduced and the erythroid cells were alsodepleted. Marked reduction in the number of megakaryocytes occurred both inthe granulocytopenic and in the granulocytopenic and anemic rats. Purpura wasnoted grossly in about 25 per cent of the rats of both groups. In anemia withoutaccompanying granulocytopenia the marrow granulocytes showed slight tomoderate depletion, whereas the erythroid index (mean) was not significantlylowered. Megakaryocytes were moderately reduced.

Lymphoid tissue—spleen, thymus, and cervical nodes—showed atrophy ofvariable degree, most marked in the thymus.

Adrenal glands showed marked depletion of cortical lipoids and rarely hemorrhage and necrosis.

Following treatment with combined folic acid, pantothenic acid, and niacinamide, granulocytopenic rats responded by showing a prompt rise in lymphocyteand polymorphonuclear leukocyte count, marked granulocyte response of thebone marrow, increased splenic hematopoiesis, lymphoid hyperplasia, and increased lipoid content of the adrenal glands.

     

Haematopoiesis in Busulphan-Treated Mice: A Comparison between Two Different Stem Cell Assays

The effect of busulphan on haematopoiesis was investigated in mice. Bone marrow and blood cells were sampled 2 h to 55 d after a single treatment with busulphan. The effect on progenitor cells was examined with the diffusion chamber (DC) technique and the spleen colony assay. Busulphan had a severe depressive effect on progenitor cells in bone marrow, and their number was reduced to 0.01–0.1 % of the control value on day 5–6. During the first 2 d the CFUs were reduced more than granulocyte progenitors as determined in the DC system. Histological examination of spleen colonies showed that in the same interval erythroid colony number was more depressed than granuloid colony number. This situation reversed after 4–6 d. Our interpretation is that busulphan affects all types of progenitor cells irrespective of their proliferative state. However, multipotent CFUs in G_o-phase are more depressed than granulocyte progenitor cells (DC), which have a higher proliferative rate. The selective effect on erythroid colonies during the initial period might indicate that busulphan impairs the ability of multipotent CFUs to erythroid differentiation. This damage was repaired within a few days. An abortive regeneration of bone marrow cells was observed on day 7–10. This can best be explained by an inflow from a cell pool intermediate between stem cells and early precursor cells, with some degree of ‘stem-cellness' (self-sustaining capacity).     

Kinetics of erythropoiesis in the liver induced in adult mice by phenylhydrazine

Phenylhydrazine treatment of normal mice elicited a rise in the numbers of CFU-S in blood, spleen and liver. High numbers of CFU-S were found in blood and liver 4 d after the first phenylhydrazine injection. CFU-S in the liver decreased slowly and were absent after 2 weeks. Blood CFU-S returned to normal levels by day 6, whereas spleen CFU-S numbers remained high upto day 12 with a 20-fold increase being apparent between days 5 and 8. Bone marrow CFU-S numbers were relatively unaffected except for a dip between days 4 and 7 with a nadir at day 5 where numbers decreased to 50% of the control levels. Approximately 40% of liver, spleen and blood CFU-S present on the 4th d after initiation of phenylhydrazine treatment, were killed with a single dose of hydroxyurea whereas bone marrow CFU-S numbers were not significantly reduced by the drug. Splenectomy performed before (21 d) or during phenylhydrazine treatment did not diminish the number of CFU-S found in theliver on day 4. A 3 d interval was observed between peak numbers of CFS-U and erythroblasts in the liver which suggests that hepatic CFU-S are able to undergo differentiation along the erythroid pathway. The presence of maceophages was correlated with that of erythroblasts in the hepatic central veins. These macrophages may be essential to the liver environment for induction of erythropoiesis.     

Erythropoietin induces the expansion of c-kit+ progenitors for myeloid and erythroid cells, but not for lymphoid cells, in the bone marrow and liver

In humans, the numbers of erythrocytes and granulocytes, but not that of lymphocytes, tend to increase in parallel. To determine the mechanism, we investigated how the administration of erythropoietin induces the expansion of erythroid cells and other lineage cells in the bone marrow, liver, and other organs of mice. When mice were injected twice (days 1 and 2) with erythropoietin at a dose of 20 or 200 IU/day/ mouse, a prominent expansion of TER 19+ (erythroid cells) and Gr-1high cells (granulocytes) occurred in the liver, spleen, and bone marrow day 3 after the initial injection. On the other hand, lymphoid cells, including NK cells, extrathymic T cells, and conventional T cells, did not expand. In parallel with the expansion of erythroid cells and granulocytes, the levels of c-kit(+)Lin- cells increased in the liver and bone marrow. Despite the increase in the proportion of c-kit(+) Lin(-) cells, the generation of lymphocytes (e.g., T cells) decreased when such bone marrow cells were injected to scid mice. These results suggest that erythropoietin has the ability to induce the expansion of not only erythroid cells but also granulocytes in the liver, spleen, and bone marrow. Furthermore, c-kit+ progenitors which may commit themselves to erythroid and myeloid cells, but not to lymphoid cells, were also activated in the liver and bone marrow of mice treated with erythropoietin.     

Stem Cell Migration Induced by Erythropoietin or Haemolytic Anaemia: the Effects of Actinomycin and Endotoxin Contamination of Erythropoietin Preparations

The injection of erythropoietin or the induction of anaemia with phenylhydrazine leads to changes in murine pluripotent and granulocyte-macrophage stem cells indicating migration from marrow to spleen. In order to evaluate the interrelationship between erythroid differentiation and stem cell migration we have selectively suppressed erythroid differentiation with actinomycin D. Anaemia or EP injection resulted in stem cell changes consistent with migration; actinomycin blocked these changes in anaemic but not EP injected mice while blocking erythropoiesis in both groups. The erythropoietin contained from 0.01 to 1000 microgram/ml of endotoxin as defined by the limulus test; it decreased marrow erythropoiesis and stimulated marrow granulopoiesis. Adsorption of the erythropoietin preparation with limulus lysate removed endotoxin without decreasing erythropoietin activity. Adsorbed erythropoietin stimulated erythropoiesis and not granulopoiesis, and stem cell changes induced by its administration were largely blocked by actinomycin, suggesting that endotoxin in the non-adsorbed erythropoietin caused the actinomycin resistant stem cell changes. The observation that actinomycin blocks both erythroid differentiation and stem cell migration suggests that these two physiologic events are closely linked. The effects of injected erythropoietin on murine haemopoietic stem cells may, to a significant extent, be secondary to the presence of endotoxin in the erythropoietin preparations.     

Hematopoiesis of hereditarily asplenic-athymic (lasat) mice.

The hematopoiesis of athymic-asplenic (lasat) mice was compared with that of normal, asplenic, and athymic littermates with the same strain background. Erythrocyte blood volume, number and survival time were normal when related to the body weight of the animals. Peripheral blood showed leukopenia with absolute and relative lymphopenia, resembling the athymic rather than the asplenic pattern. The bone marrow was hypocellular as a consequence of a decrease in both lymphocytes and erythroid precursors, while thrombocytopoiesis and granulcytopoiesis-monocytopoiesis were essentially normal. Although the percentile value of femoral stem cells was high, their absolute number was, in fact, reduced by 35% as a result of the bone marrow hypocellularity. When lasat bone marrow cells were injected into normal, lethally irradiated mice, a rapid erythropoietic recovery was observed, whereas the restoration of the granlocytic compartment was impaired. It was concluded that: 1) lasat mice depict a normal hematopoiesis in spite of the congenital absence of the thymus and the spleen; 2) bone marrow stem cells may be defective when administered to lethally irradiated hosts; and 3) the athymic status predominates over the asplenic one.     

Effects of actinomycin D in vivo on murine erythroid stem cells

Low-dose actinomycin D (Acto) selectively suppresses murine erythropoiesis without decreasing erythropoietin (Ep) production. We used the plasma clot system to determine the stage of erythroid differentiation at which this inhibition occurs. Late erythroid precursors, CFU-E, and less differentiated committed erythroid stem cells, BFU-E, were assayed in CF1 mice given Acto 75-82 microgram/kg/day or saline subcutaneously for 5 days. We also assayed pluripotent (CFU-S) and committed granulocyte-monocyte (CFU-C) stem cells. Reticulocytes and marrow and spleen nucleated erythroid precursors were decreased by 99% in the Acto-treated mice; tibial marrow CFU-E were decreased by 97% and splenic CFU-E by 99%. Tibial BFU- E were not decreased by Acto, although there was a 66% diminution in splenic BFU-E. Acto increased tibial CFU-S, but splenic CFU-S and tibial and splenic CFU-C were unchanged. Thus Acto inhibits erythropoiesis by suppressing the ability of immediate committed erythroid precursors of CFU-E or CFU-E themselves to differentiate further in response to Ep. Acto does not affect survival or proliferation of the less differentiated cells--CFU-C, CFU-S, and marrow BFU-E. The suppression of splenic BFU-E in Acto-treated mice may indicate that marrow and splenic BFU-E are basically different stem cells. Alternatively, Acto treatment may impair migration of BFU-E from marrow to spleen.     

Stem Cell Migration and Proliferation During Severe Anemia

The pluripotential stem cell (CFU) compartment of marrow and spleen wasevaluated in mice subjected to an intense erythroid stimulus associated withphenylhydrazine-induced anemia. Erythroid hyperplasia occurred in both marrow and spleen. CFU in the marrowgradually declined to approximately 50per cent of control levels (day 5) whiletheir numbers in the spleen increased(fourfold) by day 3 and were maintainedat this level for several days. Thesechanges in numbers of marrow andsplenic CFU were not associated withCFU proliferation. Thereafter, CFU inthe marrow, but not in the spleen, entered active cell cycle. The data suggestthat CFU migrate from marrow to spleenduring the demands of severe anemia.The induction of marrow CFU into cyclefurther suggests a negative feedback,which, perhaps through cell-cell interaction, maintains stem cells at a criticalcompartment size. The failure of splenicCFU to cycle may reflect the converseeffect, i.e. an inhibition on stem cell proliferation in the wake of an expandedstem cell pool.

Submitted on March 17, 1970 Revised on May 14, 1970 Accepted on June 9, 1970     

Organ specificity of c-kit+ lymphoid precursors in the liver, thymus, and bone marrow

c-kit+Lin- cells are present in various immune organs, including the liver, thymus, and bone marrow, where lymphoid, myeloid, or erythroid cells are generated. To compare their properties as lymphoid precursors, c-kit+Lin- cells purified from various organs of B6.Ly5.1 mice were injected into 6.5 Gy-irradiated B6.Ly5.2 mice. Depending on the source of the c-kit cells, the degree of entrance and expansion of lymphoid cells differed in the liver and thymus of recipient mice. c-kit+ cells isolated from the bone marrow entered and expanded prominently in both the liver and thymus, whereas c-kit+ cells from the thymus did not do so at all. On the other hand, c-kit+ cells isolated from the liver and spleen showed an intermediate pattern, namely, they took a long time to enter and expand in the liver and thymus of recipient mice. All of these c-kit+ cells had the potential to give rise to lymphoid cells, which were specific to the liver and thymus, respectively. We previously showed that progenitor cells for extrathymic T cells in the liver and those for conventional T cells in the thymus are not always supplied by the bone marrow, as shown by experiments using parabiosis. Taken together with those previous data, the present results suggest that c-kit+Lin- cells isolated from various immune organs have organ specific properties.     

The erythropoietic effects of interleukin 6 and erythropoietin in vivo

The purpose of this investigation was to compare the erythropoietic effects of recombinant interleukin 6 (IL-6) and recombinant erythropoietin (EPO) on the marrow and peripheral blood in vivo. IL-6 administered to rats as a single i.v. injection induces a selective erythroid hyperplasia of the marrow's late normoblasts at 12 and 24 h with a return to preinjection numbers of normoblasts at 48 and 72 h. The hyperplasia of late normoblasts in the marrow is accompanied by a left-shifted peripheral reticulocytosis. Daily injection of IL-6 does not induce any effects on the erythroid population of the marrow or circulation beyond those of a single injection. After daily administration of IL-6 for 4 or 7 days, the erythroid differential in the marrow and the peripheral reticulocyte count are equal to negative control values, indicating a rapid tachyphylaxis to the erythropoietic effect of IL-6. In contrast to IL-6, EPO administered as a single i.v. injection induces a panerythroid marrow hyperplasia with sequential peak increases in pronormoblasts and early normoblasts at 24 h, intermediate normoblasts at 24-48 h, and late normoblasts at 72 h. The peripheral reticulocyte count mirrors the development of erythroid precursors in the marrow by demonstrating an increasing left-shifted reticulocytosis between 24 and 72 h. Daily injection of EPO for 7 days induces a striking erythroid hyperplasia and a myeloid hypoplasia in the marrow. In summary, IL-6 in vivo is a differentiation factor that rapidly induces tolerance to its own effect, whereas EPO in vivo affects all stages of erythropoiesis and sustains erythropoiesis indefinitely. IL-6 may be one of the non-EPO factors in pokeweed mitogen spleen cell-conditioned medium that has been reported by previous investigators to enhance erythropoiesis, although many of those factors were thought to act upon an earlier stage of erythropoiesis. IL-6 is unlikely to exert an indirect erythropoietic effect in vivo via the induction of EPO because the sera of IL-6-treated rats did not contain elevated levels of EPO and because the effects of exogenously administered IL-6 and EPO are so different.     

Hematologic effects of stem cell factor in vivo and in vitro in rodents.

Recombinant rat stem cell factor (rrSCF) administered to rats as a single intravenous injection causes a dose-dependent neutrophilia and lymphocytosis as well as the appearance of immature myeloid cells and occasional blast cells in the circulation. Neutrophilia begins at 2 hours, peaks at 4 to 6 hours, and subsides between 12 and 24 hours. Lymphocytosis occurs at 0.5 hours and has subsided by 2 hours. rrSCF-induced neutrophilia and lymphocytosis are abrogated by boiling, demonstrating that endotoxin-contamination of the rrSCF preparation is not responsible for the observed hematologic effects. The bone marrow at 6 hours after injection of rrSCF shows a left-shifted myeloid and erythroid hyperplasia as evidenced by significant increases in the absolute numbers of morphologically recognizable early myeloid and erythroid precursors. A concurrent decrease in the absolute numbers of mature marrow neutrophils is noted, suggesting that the release of marrow neutrophils contributes to the peripheral neutrophilia. After 2 weeks of daily injections of rrSCF, bone marrow smears demonstrate a remarkable mast cell hyperplasia accompanied by a decrease in total marrow cellularity and by a striking erythroid and lymphoid hypoplasia. rrSCF also causes mast cells to appear in the circulation and causes a systemic increase in embryonic connective tissue-type, but not mucosal-type, mast cells. In vitro long-term culture of lineage-depleted mouse bone marrow cells with rrSCF results in an almost pure outgrowth of mast cells.     

Some Factors Involved in the Effect of X-Irradiation on the Phosphatase Activity of Hematopoietic Tissues

Factors which modify lymphoid distribution of tissues were found to modifythe adenosine triphosphatase activity of these tissues. Starvation or cortisoneinjection, which produces destructive changes in lymphoid tissues, was foundto increase the enzyme activity of spleen and thymus tissues. The greater increment of enzyme activity of the thymus as compared to that of the spleenwas correlated with its normally higher content of lymphoid tissue.

The increase in adenosine triphosphatase activity of hematopoietic tissuesappears to be associated with the type of cells present in the assay medium.With respect to peripheral blood leukocytes of the rat, the cell type is confined largely to lymphocytes and granulocytes. The increase in adenosinetriphosphatase activity of the leukocytes after total-body x-ray was seen toparallel the increase in granulocytes present in the assay medium. The ratioof granulocytes to lymphocytes is not appreciably altered in dog peripheralblood after exposure to total-body x-ray; the adenosine triphosphatase activitysimilarly was not significantly altered. After total-body x-ray (390 r and 780 r),cells isolated from the rat bone marrow displayed a fivefold increase inadenosine triphosphatase activity. This increase was seen to correspond withan increase in the ratio of segmented leukocytes and reticuloendothelial cellsand a decrease in the immature forms of the erythroid and myeloid cells.

The heterogeneous cell mixtures used for our assay procedures permit theobservation that total-body x-irradiation results in an increased enzyme activityof the isolated cells of the peripheral blood, bone marrow and spleen tissue ofthe rat. The increased enzyme activity was associated with the increased ratioof cells with high enzyme activity present in the assay medium.

Submitted on September 22, 1958 Accepted on November 27, 1958     

Mast-cell precursors in various haematopoietic colonies of mice produced in vivo and in vitro

S ummary . We have examined mast-cell precursors in various in vivo and in vitro haematopoietic colonies of mice. Cells from haematopoietic colonies of (WB x C57BL/6)F₁- +/+ mouse origin were injected into the skin of the congeneic W/W^v mice which are genetically depleted of tissue mast cells. The appearance of mast-cell clusters at the injection site indicated the presence of mast-cell precursors in the injected cell suspension. More than 40% of 12 d exogenous spleen colonies and 14 d in vitro mixed colonies contained mast-cell precursors, but only a small proportion of 7 d exogenous spleen colonies, 9 d in vitro erythroid bursts or 14 d large in vitro neutrophil-macrophage colonies contained mast-cell precursors. Neither transient endogenous erythroid spleen colonies examined at the fifth day nor 7 d in vitro neutrophil-macrophage colonies contained mast-cell precursors. Among 12 d spleen colonies mast-cell precursors were more frequent in the predominantly neutrophil than the predominantly erythroid colonies but 14 d in vitro mixed colonies, in which erythroid cells were predominant, contained mast-cell precursors more frequently than 14 d in vitro neutrophil-macrophage colonies. Therefore, differentiation of mast cells seems to be independent of either the neutrophil-macrophage or erythroid cell lineages.     

Pluripotential stem cell differentiation in hemopoietic colonies.

To determine if mononuclear cells proliferating in murine hemopoietic spleen colonies were pluripotential in addition to possessing kinetic features of stem cells, we performed sequential studies of mice during their recovery from a split-dose irradiation regimen of 850 roentgens leg shielded-3-hr interval-850 roentgens leg irradiated (850R L.S. 3- L.I.). Injecting tritiated thymidine during stem cell compartment repletion 3 and 4 days after 850R L.S. 3- L.I. resulted in heavily labeled mononuclear cells resembling medium to large leptochromatic lymphocytes in the portion of spleen removed an hour after injection. The splenic remnant obtained from the same mouse 24-48 hr later contained lightly labeled erythroblasts, myeloid cells, and lymphoid cells. Grain counts suggested that erythroblasts and their precursors had undergone about four divisions, myeloid cells and their precursors two to three divisions, and lymphoid cells and their precursors two to three divisions during the 48-hr period. Similar studies in plethoric mice demonstrated the labeling of mononuclear cells on day 4 and their differentiation to myeloid and lymphoid cells by day 6. This finding confirmed that the labeled mononuclear cells were not exclusively erythroblast progenitors. On the basis of these and previous studies of post-irradiation survival and erythropoietic recovery, we conclude that these endogenous monomuclear cells, which resemble medium to large leptochromatic lymphocytes and replicate during stem cell compartment repletion, are pluripotential hemopoietic stem cells.     

Selective eradication of rat bone marrow erythroid cells by administration of cytosine arabinoside. Observations on resumption phase of erythropoiesis.

The in vivo effect of a single i.p. injection of cytosine arabinoside (250 mg/kg body weight) on the rat bone marrow is reported. Cytosine arabinoside (ara-C), a potent inhibitor of DNA-synthesis, was seen to produce selective necrosis of fast cycling bone marrow erythroblasts. The depleted erythroid marrow is replenished by stem cells which are members of the lymphoid-transitional cells compartment. An indirect correlation between the degree of erythroid cell depletion and the extent of marrow lymphocytosis was noted 6-12 h after the administration of ara-C. At 24-48 h, however, the marrow returned to its normal activity. The rat bone marrow 6-12 h following a single injection of ara-C is suggested as an experimental model for providing stem cells committed to the erythroid line.     

Proliferative response of clonal acute myelogenous leukemia cells in localized grafts of normal bone marrow stroma to in vivo stimulation of myelopoiesis.

Bone marrow from inbred Wistar/Furth (W/Fu) rats, normal and 90% replaced by acute myelogenous leukemia (AML), was transplanted, respectively, to the left and right kidneys of adult W/Fu rats. After a transient period of hypocellularity, AML grafts repopulated to pre-transplant cellularity by day 9, whereas normal bone marrow (NBM) grafts required 25 days for repopulation to pre-transplant cellularity. Grafted leukemia cells remained localized to the kidney for 17 days. In NBM grafts phlebotomy accelerated erythroid proliferation, and intrathoracic inoculation of live Escherichia coli accelerated myeloid proliferation; in AML grafts only E. coli injection increased bone marrow proliferation. There was no morphologically detectable differentiation of W/Fu AML cells. These studies provide evidence of a myelopoietic response of AML blast cells in vivo and present a transplantation technique for comparison of localized grafts of leukemic and normal bone marrow in the same animal.     

Tissue source dictates lineage outcome of human fetal CD34(+)CD38(-) cells

OBJECTIVE: The translocation from fetal liver hematopoiesis to secondary organs occurs during the second trimester of human gestation. It has been hypothesized that stem cells migrate and acquire lineage potential based on cues specific to the adopted microenvironment. We evaluated primitive hematopoietic cell populations in the fetal human to determine if lineage restriction precedes or follows translocation to sites of hematopoietic activity including thymus, spleen, bone marrow, and liver. METHODS: Sets of hematopoietic tissues from individual second-trimester human abortuses were used to compare and quantitate the lineage outcome of immunophenotypically primitive cells from each of the hematopoietic organs using ex vivo myeloid and lymphoid differentiation systems. RESULTS: Despite uniformity in immunophenotype, functional capabilities were highly restricted by the tissue of origin and alteration in the ex vivo differentiation context did not lead to a change in differentiation outcome. CONCLUSION: Translocation of primitive cells from fetal liver to tissues of mature hematopoietic activity is associated with tissue-specific, quantitative changes in differentiation potential that are unresponsive to alternative differentiation environments. These data suggest that multipotentiality is lost prior to or upon stem-cell migration in the developing human. It is not persistent with residence in a secondary hematopoietic organ.