Prolactin-producing cells differentiate from G0/G1-arrested somatotrophs in vitro: an analysis of cell cycle phases and mammotroph differentiation.

In order to analyze the relationship between cell proliferation and mammotroph differentiation, we studied a somatotrophic cell line, MtT/S. MtT/S cell is known to differentiate into PRL-producing cells in response to stimulation with insulin or insulin-like growth factor-1 (IGF-1). Double immunostaining for bromodeoxyuridine (BrdU), which labels proliferating cells, and for GH or PRL showed that most BrdU-labeled cells were GH-immunopositive, whereas considerably few PRL-positive cells were labeled with BrdU. This was confirmed by immunostaining of proliferating cells with antibody to proliferating cell nuclear antigen (PCNA). Furthermore, flow-cytometry analysis indicated that most of the PRL-producing cells were in the G0/G1 phase of the cell cycle. In order to determine whether cell cycle changes are required for transdifferentiation of PRL-producing cells, MtT/S cells were cultivated in serum restricted medium for 7 days to reduce their mitotic activity and then treated with insulin and epidermal growth factor (EGF). Under these conditions, the cell cycle of MtT/S cells was significantly delayed, but the percentage of PRL-producing cells induced was almost identical to that under control conditions, showing that mitosis is not required for PRL- producing cell differentiation. We also labeled MtT/S cells with BrdU for 24 h during PRL-producing cell induction by insulin and EGF, and as a result BrdU-labeled proliferative cells were specifically absent from PRL-producing cell populations. These data, taken as whole, suggest that PRL cells differentiated from G0/G1 arrested somatotrophs and the PRL cells which appeared had their cell proliferation activity significantly declined. In conclusion, this is the first report showing the relationship cell between proliferation and differentiation of PRL cells.     

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.     

Self-renewal of hemopoietic stem cells during mixed colony formation in vitro.

Replating experiments have shown that the self-renewal of pluripotent hemopoietic stem cells can be studied in vitro by clonal analysis techniques. The number of daughter stem cells detectable in individual primary clones produced in vitro varies markedly from one clone to another. These findings are consistent with a general model of stem cell differentiation in which the choice to self-replicate or not is ultimately determined at the single-cell level by a mechanism involving a random-event component that is intrinsic to the stem cell itself. Hemopoietic stem cells were identified by their ability to generate macroscopic-sized colonies having a visible erythroid component (i.e., gross red color) in standard methylcellulose assays containing medium conditioned by pokeweed mitogen-treated spleen cells and erythropoietin. In assays of replated primary or secondary colonies, inclusion of irradiated marrow-cell feeders was found to be an additional requirement. The mixed erythroid-megakaryocyte-granulocyte nature of colonies identified simply as macroscopic and erythroid was confirmed by cytochemical stains for lineage-specific markers. Marked variation in self-renewal was a feature of marrow stem cells both before and after maintenance in flask culture, although the overall self-renewal capacity exhibited by flask-cultured cells was approximately 5-fold higher. Variation in self-renewal was not correlated with primary colony size, which also varied over a wide range (0.2-9 X 10(5) nucleated cells per colony). Variation in stem cell self-renewal has been previously associated with hemopoietic stem cell proliferation in vivo. Its persistence in vitro in assays of dilute single-cell suspensions casts doubt on the significance of microenvironmental influences in directing stem cell differentiation.     

Leukemia initiated by hemopoietic stem cells expressing the v-abl oncogene.

We report a mouse model with which to study leukemogenesis initiated by a specific genetic change introduced into a primary lymphoid-myeloid pluripotent stem cell. Fetal liver hemopoietic cells were infected with a high titer of helper-free Abelson murine leukemia virus (A-MuLV) and were used to reconstitute lethally irradiated mice. Two weeks later, progenies of a single primitive hemopoietic stem cell carrying a specifically integrated A-MuLV proviral DNA could be detected in both colony-forming units in spleen and myeloid colony-forming cells in the bone marrow. Beginning at 3 weeks after transplantation, the recipients developed elevated leukocyte counts, splenomegaly, and increase of blast cells in the peripheral blood. Multiple clones of A-MuLV-infected cells were infused into each recipient. However, in the same animal, DNA extracted from various affected organs and from factor-independent lymphoid and myeloid immortalized cells all contained an identical, specifically integrated proviral genome. The A-MuLV-infected stem cells differentiated into various lineages of hemopoietic cells. Our data show that the expression of the v-abl oncogene in a primary lymphoid-myeloid hemopoietic stem cell directly initiates leukemogenesis by stimulating factor-independent growth. The monoclonal-type disease development seen in these animals may require the occurrence of an additional genetic event.     

Factors controlling stem cell recirculation. III. Effect of the thymus on the migration and differentiation of hemopoietic stem cells.

Experiments were carried out to investigate the effect of thymectomy on the migration and differentiation of hemopoietic stem cells released from shielded parts of bone marrow after irradiation of mice with lethal doses of x-rays. In 2-3 wk after thymectomy, the rate of migration declined and the differentiation of stem cells into granulocytic colonies was inhibited. Transplantation of syngeneic thymus or lymph node cells into thymectomized mice enhanced the migration of stem cells from the bone marrow and restored the usual pathways of their differentiation.     

Analysis of differentiation of mouse hemopoietic stem cells in culture by sequential replating of paired progenitors

Blast cell colonies seen in cultures of spleen cells from 5- fluorouracil-treated mice provide a highly enriched population of primitive hemopoietic progenitors. Our recent studies of the differentiation potentials of the paired daughter cells of these progenitors showed different patterns of differentiation in the colonies produced by the separated daughter cells. In this study, we carried out sequential micromanipulation of paired progenitors followed by cytologic examinations of the colonies derived from these progenitors. Of the total 94 evaluable cultures, consisting of three or more colonies, 52 consisted of macrophage colonies and one consisted of megakaryocyte colonies. In the remaining 41 cultures, diverse combinations of colonies revealing heterogeneous compositions of cell lineages were identified. Presumptive genealogic trees of the differentiation of hemopoietic progenitors constructed for the latter group of cultures suggested that monopotent progenitors may be derived from pluripotent progenitors in two ways: (1) directly during one cell division of pluripotent cells or (2) as a result of progressive lineage restriction during successive division of the pluripotent progenitors. The results also suggested that some of the oligopotent progenitors are capable of limited self-renewal.     

Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro

The Notch signaling pathway plays a key role at several stages of T-lymphocyte differentiation. However, it remained unclear whether signals induced by the Notch ligand Delta-like 1 could support full T-cell differentiation from a defined source of human hematopoietic stem cells (HSCs) in vitro. Here, we show that human cord blood-derived HSCs cultured on Delta-like 1-expressing OP9 stromal cells undergo efficient T-cell lineage commitment and sustained T-cell differentiation. A normal stage-specific program of T-cell development was observed, including the generation of CD4 and CD8 alpha beta-T-cell receptor (TCR)-bearing cells. Induction of T-cell differentiation was dependent on the expression of Delta-like 1 by the OP9 cells. Stimulation of the in vitro-differentiated T cells by TCR engagement induced the expression of T-cell activation markers and costimulatory receptors. These results establish an efficient in vitro coculture system for the generation of T cells from human HSCs, providing a new avenue for the study of early T-cell differentiation and function.     

Pluripotential hemopoietic stem cells in adult mouse brain.

Single cell suspensions of adult mouse brain were shown to contain large numbers of pluripotential hemopoietic stem cells as detected by the ability to form hemopoietic colonies in the spleens of irradiated hosts. These colony forming unit, spleen (CFU-s) cells derived from brain gave rise to colonies identical in morphology and histology to those of bone marrow-derived CFU-s. The average number of CFU-s obtained per 10(5) dissociated adult brain cells was 14, whereas other adult tissues such as lung, kidney, heart, and thymus contained insignificant CFU-s levels when tested. As the level of CFU-s in adult blood is less than 1 per 10(6) nucleated cells, blood contamination does not contribute to the high levels found in adult brain. Individual spleen colonies isolated from irradiated CBA (H-2k) recipients injected with (BALB/c x CBA)F1 (H-2d x H-2k) brain cells were shown by immunofluorescence to contain cells bearing surface H-2d molecules, thus indicating that the colonies arose from the brain cell inoculum and were not endogenously derived. The surface phenotype of brain- and bone marrow-derived CFU-s was found to differ in that brain CFU-s could be inhibited by prior incubation with a monoclonal antibrain antibody B2A2, whereas bone marrow CFU-s were not. Further differences were found between brain and bone marrow CFU-s in the congenitally anemic Wf/Wf mice. These mice were shown to have a very few CFU-s in the adult bone marrow, whereas the brain contained normal adult levels. The large number of hemopoietic stem cells in the brain may indicate an essential requirement for the continual generation of cells such as microglia or phagocytic cells, without the disruption of the blood-brain barrier.     

Surface antigens of murine hemopoietic stem cells. I. Cross reactivity of antisera against differentiated hemopoietic cells with bone marrow stem cells

A model of multiply marked hemopoietic stem cells proposed by Till (1) has been tested with respect to antisera raised against differentiated murine hemopoietic cells. When absorbed with erythrocytes, antisera against CBA mouse lymph node lymphocytes, thymocytes, peritoneal macrophages and platelets cross-reacted strongly with pluripotent stem cells (CFUs) in bone marrow as determined by inhibition of spleen colony formation in lethally irradiated mice. Absorption of ATS, antimacrophage serum and antiplatelet serum with hemopoietic cells other than those used to prepare the antisera (e.g., ATS with neutrophils and platelets, antimacrophage serum with neutrophils, thymocytes and platelets and antiplatelet serum with neutrophils and thymocytes) did not reduce the activity of these antisera for CFUs whereas absorption with the inoculating cell type greatly reduced anti-stem cell activity. Absorption of these antisera with non-hemopoietic tissues such as brain, kidney, liver and testis in general had little effect on antistem cell activity, although a significant loss of activity was observed following absorption of antiplatelet serum with kidney. The antistem cell activity in ATS, antimacrophage serum and antiplatelet serum does not appear to be caused by antibodies against histocompatibility antigens sine bone marrow stem cells from histoincompatible C57BL and Balb/c mice were also sensitive to antisera against CBA mouse hemopoietic cells. In contrast to these findings, antisera against erythrocytes showed little cross-reactivity with CFUs, indicating that few antigens are held in common between erythrocytes and CFUs. We propose that nucleated hemopoietic cells and platelets retain cell line specific antigens in common with pluripotent stem cells from which they were derived, and that the continued expression of these antigens during differentiation may be involved in the differentiation process.     

In vitro generation of hematopoietic stem cells from an embryonic stem cell line.

Hematopoietic stem cells (HSC) are unique in that they give rise both to new stem cells (self-renewal) and to all blood cell types. The cellular and molecular events responsible for the formation of HSC remain unknown mainly because no system exists to study it. Embryonic stem (ES) cells were induced to differentiate by coculture with the stromal cell line RP010 and the combination of interleukin (IL) 3, IL-6, and F (cell-free supernatants from cultures of the FLS4.1 fetal liver stromal cell line). Cell cytometry analysis of the mononuclear cells produced in the cultures was consistent with the presence of PgP-1+ Lin- early hematopoietic (B-220- Mac-1- JORO 75- TER 119-) cells and of fewer B-220+ IgM- B-cell progenitors and JORO 75+ T-lymphocyte progenitors. The cell-sorter-purified PgP-1+ Lin- cells produced by induced ES cells could repopulate the lymphoid, myeloid, and erythroid lineages of irradiated mice. The ES-derived PgP-1+ Lin- cells must possess extensive self-renewal potential, as they were able to produce hematopoietic repopulation of secondary mice recipients. Indeed, marrow cells from irradiated mice reconstituted (15-18 weeks before) with PgP-1+ Lin- cell-sorter-purified cells generated by induced ES cells repopulated the lymphoid, myeloid, and erythroid lineages of secondary mouse recipients assessed 16-20 weeks after their transfer into irradiated secondary mice. The results show that the culture conditions described here support differentiation of ES cells into hematopoietic cells with functional properties of HSC. It should now be possible to unravel the molecular events leading to the formation of HSC.     

Survival of hemopoietic progenitors in the G0 period of the cell cycle does not require early hemopoietic regulators.

Although it is generally held that hemopoietic stem cells in steady-state marrow are dormant in the cell cycle, the direct proof for this concept has been lacking. In the present study, we have documented the development of human multipotential blast cell colonies from single cells by daily observation of the growth of candidate progenitors. The results clearly demonstrated that early hemopoietic progenitors may remain as single cells for more than 2 weeks of incubation. Once the progenitors began proliferation, the subsequent growth was characterized by steady cell doubling. Next, we tested the survival of blast cell colony progenitors in the presence of neutralizing antibodies prepared against early acting hemopoietic factors including interleukin (IL) 1 alpha, IL-1 beta, IL-3, IL-6, and granulocyte colony-stimulating factor. Cultures were initiated with individual antibodies, and, on day 14, IL-3 and the corresponding growth factor in concentrations that neutralize the antibodies were added. On days 18-27 of culture, blast cell colonies containing 25 or more cells were identified and replated for analysis of their ability to form secondary colonies. The cumulative frequency of the blast cell colonies in cultures containing antibody did not differ significantly from that of the control group containing rabbit IgG. A combination of anti-IL-1 alpha, anti-IL-1 beta, anti-IL-6, and anti-granulocyte colony-stimulating factor did not affect the survival of dormant blast cell colony-forming cells. These results indicate that survival of hemopoietic stem cells in the G0 period of the cell cycle is independent of early hemopoietic regulators.     

Demonstration of hemopoietic stem cells in the peripheral blood of baboons by cross circulation.

Baboons were given 1200 R total body irradiation from two opposing 60Co sources. Three animals were given supportive therapy only and died, as expected, within 8 days of irradiation with profound marrow hypoplasia. Five baboons were cross-circulated with unirradiated partners and died within 14 days with evidence suggestive of graft-versus-host disease. Their marrows were repopulated with hemopoietic precursor cells, and three of the five had rises in peripheral white blood cell counts to more than 1500/cu mm before death. These results are compatible with the presence of hemopoietic stem cells in the peripheral blood of a nonhuman primate, the baboon.     

Intrinsic potential of phenotypically defined human hemopoietic stem cells to self-renew in short-term in vitro cultures.

In search for culture conditions that will facilitate hemopoietic stem cell (HSC) replication while preserving their primitive properties, we have made use of a multi-parameter FACS assay to define HSCs on basis of their phenotypic characteristics, i.e., CD34++CD33,38,71(-). Bone marrow and umbilical cord blood samples of CD34(+) cells from 31 donors were loaded with the membrane dye PKH26 and each exposed to various culture conditions for 6 days. The cells that retained the primitive CD34(++)CD33,38,71(-) phenotype were analysed for the number of cell replications they underwent, by measuring loss of PKH26 fluorescence after 6 days. A most striking observation was the large inter-sample variation in the proliferative response of cells that retained the CD34(++)CD33,38,71(-) phenotype. In general, samples could be characterised as either good- or poorly-replicating, according to the proliferation property of their CD34(++)CD33,38,71(-) subset. In comparison to this 'intrinsic' potential, the effects of the applied growth stimuli on CD34(++)CD33,38,71(-) cell replication were negligible. In contrast, the overall recovery of the CD34(++)CD33,38,71(-) cells was clearly dependent on the culture stimuli. Of the various conditions tested, serum-free cultures with pre-established stroma maintained the cells with this primitive phenotype most effectively. In cultures supplemented with various combinations of recombinant HGFs, HSC differentiation prevailed. These findings with phenotypically defined HSCs should assist in the design of systems for expansion and ex vivo gene therapy of early hemopoietic cells.     

Allogeneic hemopoietic stem cell transplantation using mouse spleen cells fractionated by lectins: in vitro study of cell fractions.

Mouse spleen cells sequentially agglutinated by soybean agglutinin (SBA) and peanut agglutinin (PNA) previously had been shown to be sufficiently depleted of graft-versus-host activity to allow reconstitution of lethally irradiated allogeneic recipient mice. We have now tested the extent of T-cell depletion in this cell fraction by various in vitro assays, including cytotoxicity testing with anti-Thy-1 antiserum, mitogenic response to the T-cell mitogens concanavalin A and phytohemagglutinin and allogeneic responsiveness in the mixed lymphocytes culture assay. By these criteria the SBA+, PNA+ spleen fraction, used previously in the in vivo experiments, was found to possess about 1% T-cell contamination. The slight contamination with T cells previously found in the singly agglutinated SBA fraction can be removed by a second fractionation with SBA, thus eliminating the possibility that a minor T-cell subpopulation bears receptors for SBA. Finally, we demonstrated that the twice-agglutinated fraction, by SBA and PNA or by SBA alone, contains a significant number of prothymocytes, thereby indicating that mouse prothymocytes bear receptors for both SBA and PNA. The implication of these findings to bone marrow transplantation in humans is discussed.     

In vitro homing of hemopoietic stem cells is mediated by a recognition system with galactosyl and mannosyl specificities.

We synthesized a number of neoglycoprotein probes by covalently linking three biologically relevant sugars (mannose, galactose, and fucose) to a protein molecule so as to retain the pyranose (ring) form of sugars necessary for their interaction with lectins. In the presence of galactosyl and mannosyl but not fucosyl probes, the production of CFU-S [colony-forming unit(s) in spleen] and total cells was halted in murine long-term marrow cultures. Cobblestone areas disappeared in these cultures, indicating the inhibition of binding of hemopoietic cells to the stroma. Electron microscopy revealed no alterations of the stroma, and the probes did not have direct cytotoxic or inhibitory effects on the growth of CFU-S or CFU-C [colony-forming unit(s) in culture]. Stroma grown for 5 weeks in the presence of the probes could subsequently support the growth of hemopoietic progenitor cells when the probes were removed from the medium. Conversely, the proliferative capacity of CFU-S in the supernate, grown in the presence of the probes, was retained upon grafting to control stroma. Galactosyl and mannosyl but not fucosyl probes differentially agglutinated CFU-S in whole-marrow-cell suspensions, suggesting the presence of membrane lectins with specificity for these sugars on the surface of CFU-S. We conclude that the binding of CFU-S to marrow stroma (homing) is mediated by a recognition system with galactosyl and mannosyl specificities.     

Interleukin-3 and granulocyte colony-stimulating factor as survival factors in murine hemopoietic stem cells in vitro.

We examined the role of various hemopoietic factors in the survival of hemopoietic stem cells in methylcellulose culture. Bone marrow cells from 5-fluorouracil (5-FU)-treated mice were cultured without hemopoietic factors. Several days later, a mixture of colony-stimulating factors (CSF interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), and erythropoietin (Ep)) was added to the culture (delayed addition of CSF) to induce the maximal colony growth in surviving progenitors. In this system few colonies grew, suggesting that some hemopoietic factors are required for the survival of hemopoietic stem cells in vitro. In a further series of experiments, similar cultures were initiated with single known hemopoietic factors or with a mixture of CSF, followed by the addition of CSF 7 days later. Although IL-3 and G-CSF, as single factors, supported colony growth, the other factors did not. In this experiment, while the total number of colonies in cultures initiated with IL-3 or G-CSF was less than that observed in cultures initiated with a mixture of CSF, the number of multipotential GEMM (granulocyte-erythrocyte-macrophage-megakaryocyte) colonies remained constant. We concluded that IL-3 and G-CSF played important roles as single factors in the survival of murine dormant hemopoietic stem cells in vitro.     

Growth and differentiation of circulating hemopoietic stem cells with atomic bomb irradiation-induced chromosome abnormalities

The effects of atomic bomb irradiation on hemopoietic stem cells were studied cytogenetically using single colonies derived from hemopoietic progenitor cells. The subjects studied were 21 healthy atomic bomb survivors (10 males and 11 females) in the high dose exposure group (100+ rad) with a known high incidence (10% or more) of radiation-induced chromosome abnormalities in their peripheral blood lymphocytes (stimulated with phytohemagglutinin), and 11 nonexposed healthy controls (5 males and 6 females). Colony formation by circulating granulocyte-macrophage (GM-CFC) and erythroid (BFU-E) progenitor cells was made by the methylcellulose method using peripheral blood mononuclear cells. Chromosome specimens were prepared from single colonies by our micromethod. The total number of colonies analyzed in the exposed group was 131 for GM-CFC and 75 for BFU-E. Chromosome abnormalities were observed in 15 (11.5%) and 9 (12.0%) colonies, respectively. In the control group, the total number of colonies analyzed was 61 for GM-CFC and 41 for BFU-E. None of these colonies showed chromosome abnormalities. The difference in incidence of chromosome abnormalities was highly significant by an exact test; p = 0.003 for GM-CFC and 0.017 for BFU-E. The karyotypes of chromosome abnormalities obtained from the colonies in the exposed group were mostly translocations, but deletion and marker chromosomes were also observed. In two individuals, such karyotypic abnormalities as observed in the peripheral lymphocytes were also seen in the myeloid progenitor cells. This finding suggests that atomic bomb irradiation produced a chromosome aberration on multipotent hemopoietic stem cells common to myeloid and lymphoid lineages. These stem cells, although carrying chromosome defects, are likely to have survived for more than 30 years, continuously producing progenitor cells capable of normal-looking growth and differentiation.     

Hepatic eosinophilopoiesis from multipotent hemopoietic stem cells in Toxocara canis-infected mice

Extramedullary hemopoiesis, recognized as hemopoietic foci, increased in the livers of Toxocara canis-infected mice. At the peak of the response (day-13 after infection), the majority of hepatic hemopoietic foci were of the eosinophil lineage. Hepatic nonparenchymal cells prepared from T. canis-infected mice on day 13 contained large numbers of hemopoietic stem cells, more than half of which were cycling. When W/Wv mice, which are genetically deficient in multipotent hemopoietic stem cells, were infected with T. canis, hepatic hemopoietic foci were rare throughout the course of infection. This impaired response of W/Wv mice was restored by bone marrow grafting from normal +/+ littermates. These results indicate that, in response to the increased demand, eosinophils are generated in the liver by the differentiation from multipotent stem cells, not only from the committed precursors.