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PubMed2005

Stem cells are units of natural selection in a colonial ascidian.

Stem cells are highly conserved biological units of development and regeneration. Here we formally demonstrate that stem cell lineages are also legitimate units of natural selection. In a colonial ascidian, Botryllus schlosseri, vascular fusion between genetically distinct individuals results in cellular parasitism of somatic tissues, gametes, or both. We show that genetic hierarchies of somatic and gametic parasitism following fusion can be replicated by transplanting cells between colonies. We prospectively isolate a population of multipotent, self-renewing stem cells that retain their competitive phenotype upon transplantation. Their single-cell contribution to either somatic or germline fates, but not to both, is consistent with separate lineages of somatic and germline stem cells or pluripotent stem cells that differentiate according to the niche in which they land. Since fusion is restricted to individuals that share a fusion/histocompatibility allele, these data suggest that histocompatibility genes in Botryllus evolved to protect the body from parasitic stem cells usurping asexual or sexual inheritance.

Animals↗
PubMed2005

Efficiency of transduction of highly purified murine hematopoietic stem cells by lentiviral and oncoretroviral vectors under conditions of minimal in vitro manipulation.

The development of leukemias in several children with severe combined immunodeficiency disease who were transplanted with retroviral vector-transduced bone marrow cells has renewed concerns about the risks associated with the random integration of proviral sequences into chromosomal DNA. One theoretical way to reduce the risks of insertional mutagenesis would be to employ transduction/transplantation protocols that minimize the total number of genetically modified cells and associated proviral integration "events" introduced into recipients. Toward this end, we have developed a transduction protocol that involves the short-term incubation of highly purified murine stem cells with high-titer recombinant lentivirus vectors in the presence of serum-free medium and the cytokines SCF and TPO. Competitive repopulation studies showed that stem cells transduced in this way possessed the same reconstitutive ability as fresh, unmanipulated cells. Animals transplanted with only 200-2000 transduced cells were efficiently reconstituted with the genetically modified cells, and most hematopoietic cells in the recipients expressed the transgene. In contrast, the use of high-titer oncoretroviral vectors in conjunction with the same transduction/transplantation protocol resulted in only low levels of gene marking in vivo. The use of a similar transduction/transplantation strategy in future clinical studies may offer distinct advantages over current protocols.

Animals↗
PubMed1999

Engraftment and multilineage expression of human bone marrow CD34- cells in vivo.

The fetal sheep competitive engraftment model of human hematopoietic stem cells (HSC) was used to evaluate the in vivo engraftment potential of human bone marrow CD34- Lin- cells. Transplantation of CD34- Lin- cells into primary hosts resulted in the long-term (> 1 year) engraftment and multilineage donor cell/progenitor expression with production of significant numbers of CD34+ cells. Secondary transplantation and limiting dilution studies confirmed the presence in human CD34- fraction of HSC with in vivo long-term engraftment and multilineage differentiation potentials.

Animals↗
PubMedDate not supplied

[Hematopoietic stem cells].

The hematopoietic system is the adult cellular model in which the biology of the stem cells is the best known and may be a model for numerous other tissues. This model is theoretically based on a hierarchy of cells, which begin on a stem cell that differentiates into mature cells through a large number of cellular stages including hematopoietic progenitors. Hematopoietic stem cells have three main properties: (1) Self-renewal capacities. However, in transplantation experiments this property is limited and may be regulated by the stem cell niche. (2) Multipotentiality. Hematopoietic stem cells are capable of differentiation towards all myeloid and lymphoid lineages. However, recent experiments suggest that, like other somatic stem cells, hematopoietic stem cells are capable of differentiating towards non-hematopoietic lineages. The mechanisms of call commitment towards one cell lineage are beginning to be understood and involve competition between transcription factors. (3) Migration. Stem cells are able to travel through the blood. These migration capacities may be an important but underestimated role in the function of hematopoietic stem cells. Mobilization of stem cells by growth factors is now clinically used for hematopoietic transplantation in man. CXCR4 and its ligand, the SDF-1 chemokine, play an important role in stem cell chemotaxis and in their retention in the marrow.

Animals↗
PubMed2005

Quiescence of hematopoietic stem cells and maintenance of the stem cell pool is not dependent on TGF-beta signaling in vivo.

OBJECTIVE: Maintained quiescence of hematopoietic stem cells (HSCs) is of critical importance to prevent premature exhaustion of the stem cell pool under conditions of hematopoietic stress. The growth inhibitory cytokine transforming growth factor beta (TGF-beta) has been shown to play a critical role in maintaining quiescence of HSCs in vitro. Here, we have used conditional knockout mice for the TGF-beta type I receptor (TbetaRI) to ask whether the naturally quiescent state of HSCs in vivo is dependent on TGF-beta signaling and thus whether TGF-beta serves as a protective factor for the stem cell pool during conditions of stress. METHODS: TbetaRI null and control bone marrow chimeras were subjected to repeated treatments with the cell cycle-specific cytotoxic drug 5-fluorouracil (5-FU) and surviving HSCs were assayed by competitive transplantation experiments. Exhaustion of stem cells was provoked by serially transplanting TGF-beta signaling-deficient as well as normal BM cells into lethally irradiated recipients, which were monitored for survival. RESULTS: Surprisingly, we found that TGF-beta receptor-deficient HSCs have similar susceptibility, compared to controls, to repeated 5-FU treatments, indicative of normally maintained quiescence in these cells. Likewise, hematopoietic failure occurred at similar stages in serially transplanted recipients of TbetaRI null and control BM, respectively, demonstrating normal consumption of the stem cell pool during hematopoietic stress. CONCLUSIONS: These findings clearly demonstrate that, despite a key role in vitro, TGF-beta does not provide the necessary signal that induces the quiescent state of HSCs and maintains the stem cell pool in vivo.

Animals↗
PubMed2000

An in vivo competitive repopulation assay for various sources of human hematopoietic stem cells.

The marrow repopulating potential (MRP) of different sources of human hematopoietic stem cells (HSCs) was directly compared using an in vivo assay in which severe combined immunodeficient disease (SCID) mice were implanted with human fetal bones. HSCs from 2 human lymphocyte antigen (HLA)-mismatched donors were injected individually or simultaneously into the fetal bones of a 3rd distinct HLA type and donor and recipient myeloid and lymphoid cells were identified after 8 to 10 weeks. The study compared the MRP of umbilical cord blood (CB) and adult bone marrow (ABM) CD34(+) cells as well as grafts of each type expanded ex vivo. Equal numbers of CB and ABM CD34(+) cells injected individually demonstrated similar abilities to establish multilineage hematopoiesis. However, when CB and ABM cells were transplanted simultaneously, the engraftment of CB cells was markedly superior to ABM. CB and ABM CD34(+) cells were expanded ex vivo using either a porcine microvascular endothelial cell (PMVEC)-based coculture system or a stroma-free expansion system. Primary CB CD34(+) cells or CD34(+) cells expanded in either culture system demonstrated a similar ability to engraft. However, the MRP of expanded grafts simultaneously injected with primary CB cells was uniformly inferior to primary CB cells. CD34(+) cell grafts expanded in the stroma-free system, furthermore, outcompeted CD34(+) cells expanded using the PMVEC coculture system. The triple HLA-mismatched SCID-hu model represents a novel in vivo stem cell assay system that permits the direct demonstration of the functional consequences of ex vivo HSC expansion and ontogeny-related differences in HSCs.

Animals↗
PubMed1980

Competitive repopulation: a new assay for long-term stem cell functional capacity.

A new assay for the long-term functional capacity of hemopoietic stem cells is reported. Stem cell function in each donor of a particular genotype is assayed by mixing its marrow cells with a constant number of marrow cells from a donor with distinguishable hemoglobin and measuring the relative ability of each donor to populate stem-cell-depleted recipients. For example, cells from many different B6 donors may be assayed by mixing them with a constant number of WBB6F1 cells from a single pool and injecting them into irradiated WBB6F1 recipients. As the ratio of B6 to WBB6F1 marrow cells increases from 0:1 to 3:1 the percentage of B6 hemoglobin increases in a linear fashion. This is also found with WB and WBB6F1 or CBA and B6CBAF1 mixtures. Correlation coefficients between the percentage of hemoglobin of one donor type and the ratio of cells in the mixture of that type ranged from 0.78 to 0.98 in single experiments, and were 0.68 and 0.75 using data pooled from several experiments.

Animals↗
PubMed1989

Clonal contributions of small numbers of retrovirally marked hematopoietic stem cells engrafted in unirradiated neonatal W/Wv mice.

Mice were repopulated with small numbers of retrovirally marked hematopoietic cells operationally definable as totipotent hematopoietic stem cells, without engraftment of cells at later stages of hematopoiesis, in order to facilitate analysis of stem cell clonal histories. This result depended upon the use of unirradiated W/Wv newborn recipients. Before transplantation, viral integration markers were introduced during cocultivation of fetal liver or bone marrow cells with helper cell lines exporting defective recombinant murine retroviruses of the HHAM series. Omission of selection in culture [although the vector contained the bacterial neomycin-resistance (neo) gene] also limited the proportion of stem cells that were virally labeled. Under these conditions, engraftment was restricted to a small population of marked and unmarked normal donor stem cells, due to their competitive advantage over the corresponding defective cells of the mutant hosts. A relatively simple and coherent pattern emerged, of one or a few virally marked clones, in contrast to previous studies. In order to establish the totipotent hematopoietic stem cell identity of the engrafted cells, tissues were sampled for viral and inbred-strain markers for periods close to one year after transplantation. The virally labeled clones were characterized as stem cell clones by their extensive self-renewal and by formation of the wide range of myeloid and lymphoid lineages tested. Results clearly documented concurrent contributions of cohorts of stem cells to hematopoiesis. A given stem cell can increase or decrease its proliferative activity, become completely inactive or lost, or become active after a long latent period. The contribution of a single clone present in a particular lineage was usually between 5% and 20%.

Animals↗
PubMed1997

Cellular mechanisms for developmental toxicity of chlorpyrifos: targeting the adenylyl cyclase signaling cascade.

Developmental neurotoxicity caused by chlorpyrifos exposure is generally thought to target cholinesterase but chlorpyrifos may also act on cellular intermediates, such as adenylyl cyclase, that serve global functions in the coordination of cell development. In the current study, neonatal rats were exposed to apparently subtoxic doses of chlorpyrifos (no weight loss, no mortality) either on Postnatal Days 1-4 or on Postnatal Days 11-14, and the effects on components of the adenylyl cyclase cascade were evaluated in brain regions that are enriched (forebrain) or sparse (cerebellum) in cholinergic innervation, as well as in a nonneural tissue (heart). In all three, chlorpyrifos evoked deficits in multiple components of the adenylyl cyclase cascade: expression and activity of adenylyl cyclase itself, functioning of G-proteins that link neurotransmitter and hormone receptors to cyclase activity, and expression of neurotransmitter receptors that act through this cascade. Disruption of signaling function was not restricted to transduction of cholinergic signals but rather extended to adrenergic signals as well. In most cases, the adverse effects were not evident during the immediate period of chlorpyrifos administration, but appeared after a delay of several days. These results suggest that chlorpyrifos can affect cell development by altering the activity and reactivity of the adenylyl cyclase signaling cascade, a major control point for trophic regulation of cell differentiation. The effects are not restricted to cholinergic targets, nor even to the central nervous system. Hence, disruption of cell development by chlorpyrifos is likely to be more widespread than previously thought.

Adenylyl Cyclases↗
PubMed1996

Primitive hematopoietic cells in murine bone marrow express the CD34 antigen.

The CD34 antigen is expressed on most, if not all, human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells, and its use for the enrichment of HSCs with repopulating potential is well established. However, despite homology between human and murine CD34, its expression on subsets of primitive murine hematopoietic cells has not been examined in full detail. To address this issue, we used a novel monoclonal antibody against murine CD34 (RAM34) to fractionate bone marrow (BM) cells that were then assayed in vitro and in vivo with respect to differing functional properties. A total of 4% to 17% of murine BM cells expressed CD34 at intermediate to high levels, representing a marked improvement over the resolution obtained with previously described polyclonal anti-CD34 antibodies. Sixty percent of CD34+ BM cells lacked lineage (Lin) markers expressed on mature lymphoid or myeloid cells. Eighty-five percent of Sca-1+Thy-1(10)Lin-/10 cells that are highly enriched in HSCs expressed intermediate, but not high, levels of CD34 antigen. The remainder of these phenotypically defined stem cells were CD34-. In vitro colony-forming cells, day-8 and -12 spleen colony-forming units (CFU-S), primitive progenitors able to differentiate into B lymphocytes in vitro or into T lymphocytes in SCID mice, and stem cells with radioprotective and competitive long-term repopulating activity were all markedly enriched in the CD34+ fraction after single-parameter cell sorting. In contrast, CD34-BM cells were depleted of such activities at the cell doses tested and were capable of only short-term B-cell production in vitro. The results indicate that a significant proportion of murine HSCs and multilineage progenitor cells express detectable levels of CD34, and that the RAM34 monoclonal antibody is a useful tool to subset primitive murine hematopoietic cells. These findings should facilitate more direct comparisons of the biology of CD34+ murine and human stem and progenitor cells.

Animals↗
PubMed2004

Competitive clonal hematopoiesis in mouse chimeras explained by a stochastic model of stem cell organization.

Many current experimental results show the necessity of new conceptual approaches to understand hematopoietic stem cell organization. Recently, we proposed a novel theoretical concept and a corresponding quantitative model based on microenvironment-dependent stem cell plasticity. The objective of our present work is to subject this model to an experimental test for the situation of chimeric hematopoiesis. Investigating clonal competition processes in DBA/2-C57BL/6 mouse chimeras, we observed biphasic chimerism development with initially increasing but long-term declining DBA/2 contribution. These experimental results were used to select the parameters of the mathematical model. To validate the model beyond this specific situation, we fixed the obtained parameter configuration to simulate further experimental settings comprising variations of transplanted DBA/2-C57BL/6 proportions, secondary transplantations, and perturbation of stabilized chimeras by cytokine and cytotoxic treatment. We show that the proposed model is able to consistently describe the situation of chimeric hematopoiesis. Our results strongly support the view that the relative growth advantage of strain-specific stem cells is not a fixed cellular property but is sensitively dependent on the actual state of the entire system. We conclude that hematopoietic stem cell organization should be understood as a flexible, self-organized rather than a fixed, preprogrammed process.

Animals↗
PubMed1984

Specific inhibition of in vitro lymphocyte transformation by an anti-pan T cell (gp67) ricin A chain immunotoxin.

The toxin A chain of ricin has been conjugated by a disulfide bond to a murine monoclonal antibody that recognizes the gp67kD antigen present on 95% of peripheral T lymphocytes. The immunotoxin retains both functions of its component parts: it binds to human peripheral blood lymphocytes, and it inhibits protein synthesis in a cellfree reticulocyte system. The immunotoxin has been evaluated for its ability to inhibit in vitro T lymphocyte transformation. In the presence of 20 mM NH4Cl, the immunotoxin decreases lymphocyte proliferation in response to phytohemagglutinin to less than 8% of untreated controls. The proliferative response in mixed lymphocyte culture and the development of allocytotoxic T cells is also dramatically inhibited by this immunotoxin. Monoclonal antibody alone does not inhibit these responses. Specificity of the immunotoxin has been established: the effect of the immunotoxin can be blocked by unconjugated monoclonal antibody, but not by a control monoclonal antibody that recognizes another T lymphocyte differentiation antigen or by a control monoclonal antibody that does not recognize human peripheral blood leukocytes. Treatment of human bone marrow cells with the immunotoxin preserves hematopoietic progenitor cells, as measured by granulocyte-macrophage, erythroid, and multipotential hematopoietic progenitor cell assays. These results indicate that an anti-pan T lymphocyte-ricin A chain immunotoxin is an effective agent against immunocompetent T lymphocytes in vitro, and may be an effective agent for use in clinical bone marrow transplantation.

Animals↗
PubMed1984

Ultimate erythropoietic repopulating abilities of fetal, young adult, and old adult cells compared using repeated irradiation.

Erythropoietic repopulating abilities of fetal liver cells and young and old adult marrow cells were compared as follows: Equal numbers of cells from a donor of each age were mixed with a constant portion of cells pooled from genetically distinguishable competitors. These mixtures were transplanted into stem cell-depleted recipients, and the proportions of recipient hemoglobin that were donor type measured the relative effectiveness of early erythropoietic precursor cells from the various donors (Fig. 1). At intervals of 3-6 mo, recipients were sublethally irradiated, requiring a new round of competitive repopulation. When B6 mice were used as donors, with WBB6F1 competitors and recipients, the highest levels of stem cell activity were found using old donors (Tables I, III). This was true even with unirradiated, immune-competent W/Wv recipients (Table III). When donors and recipients were WBB6F1 hybrids, with B6 competitors, fetal cells initially gave higher levels of repopulating ability, and they were similar to the adult and old marrow cells after 400 d and after recovery from two sublethal irradiations (Table II). These effects were mostly insignificant and probably reflect small differences in initial stem cell concentrations that are brought out by the sensitivity of the competitive repopulation assay. Clearly, ultimate erythropoietic stem cell proliferative capacities did not decline as a result of the proliferation required between 15 d of fetal life and old age. Repopulating abilities of 12-d fetal liver cells were not detectable. We also showed that the proportions of newly synthesized hemoglobins made by the two types of stem cells in tetraparental mice remained nearly constant when tested at 3-d intervals over 30 d. Minimum numbers of stem cells producing erythrocytes over a single 3-d period were calculated as 62 and 128, but these are too low, since variances were similar in the tetraparental mice and in the F1 hybrid control. This contradicts the hypothesis that erythropoietic stem cells reserve limited proliferative capacities by proliferating one or a few at a time. We suggest that erythropoietic stem cells have essentially unlimited proliferative capacities and are found in approximately equal concentrations in the primary erythropoietic organs after 15 or 16 d of fetal life.

Aging↗
PubMed1998

Improved gene transfer into baboon marrow repopulating cells using recombinant human fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor.

We have used a competitive repopulation assay in baboons to develop improved methods for hematopoietic stem cell transduction and have previously shown increased gene transfer into baboon marrow repopulating cells using a gibbon ape leukemia virus (GALV)-pseudotype retroviral vector (Kiem et al, Blood 90:4638, 1997). In this study using GALV-pseudotype vectors, we examined additional variables that have been reported to increase gene transfer into hematopoietic progenitor cells in culture for their ability to increase gene transfer into baboon hematopoietic repopulating cells. Baboon marrow was harvested after in vivo administration (priming) of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF). CD34-enriched marrow cells were divided into two equal fractions to directly compare transduction efficiencies under different gene transfer conditions. Transduction by either incubation with retroviral vectors on CH-296-coated flasks or by cocultivation on vector-producing cells was studied in five animals; in one animal, transduction on CH-296 was compared with transduction on bovine serum albumin (BSA)-coated flasks. The highest level of gene transfer was obtained after 24 hours of prestimulation followed by 48 hours of incubation on CH-296 in vector-containing medium in the presence of multiple hematopoietic growth factors (interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor). Using these conditions, up to 20% of peripheral blood and marrow cells contained vector sequences for more than 20 weeks, as determined by both polymerase chain reaction and Southern blot analysis. Gene transfer rates were higher for cells transduced on CH-296 as compared with BSA or cocultivation. In one animal, we have used a vector expressing a cell surface protein (human placental alkaline phosphatase) and have detected 10% and 5% of peripheral blood cells expressing the transduced gene 2 and 4 weeks after transplantation as measured by flow cytometry. In conclusion, the conditions described here have resulted in gene transfer rates that will allow detection of transduced cells by flow cytometry to facilitate the evaluation of gene expression. The levels of gene transfer obtained with these conditions suggest the potential for therapeutic efficacy in diseases affecting the hematopoietic system.

Animals↗
PubMed1982

Humoral regulation of erythropoiesis and thrombopoiesis in appropriate and small for gestational age infants.

Fourteen infants with birth weight appropriate for gestational age (AGA) and 16 small for gestational age (SGA) infants were investigated for haemoglobin concentration, haematocrit level and thrombocyte count on the first day of life. Cord serum was tested for erythropoietin (ESF) and thrombopoietin (TSF) activity. The same investigations were performed on venous blood and serum from 18 healthy adult individuals. SGA infants had higher haemoglobin concentration and haematocrit level (p less than 0.05), and lower platelet count (p less than 0.001) than AGA infants. Significant ESF activity was present in cord serum, but was not detectable in serum from adults. Significant TSF activity was present in cord serum as well as in serum from adults, with higher levels in the newborn infants (p less than 0.05). An inverse relationship was found between serum TSF activity and the number of platelets in adults, which was not demonstrable in newborn infants. Long-term intrauterine hypoxia because of placental dysfunction may be the reason for polycythemia and thrombocytopenia in SGA infants. Thrombocytopenia may be caused by competitive mechanisms on common stem cells for erythropoiesis and thrombopoiesis, shunting stem cells in direction of erythropoiesis during hypoxic exposure. Normal serum TSF activity in SGA infants indicates that lack of the humoral factor for platelet production is not the reason for the thrombocytopenia in these infants. A negative feed-back mechanism may exist between platelet number and TSF production.

Adult↗
PubMed1980

Transitory thrombocytopenia in newborn mice after intrauterine hypoxia.

Pregnant mice were exposed to hypobaric hypoxia (0.7 atmospheres) continuously during days 13 to 20 of pregnancy. In the newborn offspring, the body weights were decreased, and liver weight/body weight ratios were increased compared to nonhypoxic control animals (P < 0.001). After intrauterine hypoxia, the newborn animals were polycythemic, and a postnatal transitory thrombocytopenia lasting 7 days occurred. A rebound thrombocytosis appeared on days 13 to 15 after birth. Bone marrow megacaryocyte concentrations were increased in the hypoxic animals on days 5 to 15 after birth compared to the nonhypoxic controls. These findings may be explained by a competitive mechanism on common stem cells. Increased demand for erythropoiesis during hypoxia may shunt hematopoietic cells in direction of erythropoiesis at expense of thrombopoiesis.

Animals↗