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1.
The wall of the chick embryo aorta harbours M-CFC, G-CFC, GM-CFC and BFU-E   总被引:7,自引:0,他引:7  
In the 3- to 4-day avian embryo, after the first wave of haemopoiesis which derives in the yolk sac from haemopoietic stem cells formed in situ, haemopoietic cells emerge in an intraembryonic site, the wall of the aorta. In this paper, we demonstrate that this site harbours M-CFC, G-CFC, GM-CFC and late and early BFU-E. In serum-free medium, the growth of M-CFC and GM-CFC was strictly dependent on CSF present in fibroblast-conditioned medium (FCM). The growth of G-CFC was improved when FCM was replaced by a minute quantity of chicken and fetal calf serum. Like erythroid progenitors from bone marrow, BFU-E detected here required anaemic chicken serum to differentiate into haemoglobinized cells. The frequency of the different types of haemopoietic progenitors in the aortic population was very high: 80 M-CFC, 25 G-CFC, 4 GM-CFC and 70 BFU-E for 12,500 aorta cells, i.e. two to eight times more frequent than in the bone marrow population, depending on the type of progenitors.  相似文献   

2.
Colony formation in agar by multipotential hemopoietic cells.   总被引:7,自引:0,他引:7  
Agar cultures of CBA fetal liver, peripheral blood, yolk sac and adult marrow cells were stimulated by pokeweed mitogen-stimulated spleen conditioned medium. Two to ten percent of the colonies developing were mixed colonies, documented by light or electron microscopy to contain erythroid, neutrophil, macrophage, eosinophil and megakaryocytic cells. No lymphoid cells were detected. Mean size for 7-day mixed colonies was 1,800-7,300 cells. When 7-day mixed colonies were recloned in agar, low levels of colony-forming cells were detected in 10% of the colonies but most daughter colonies formed were small neutrophil and/or macrophage colonies. Injection of pooled 7-day mixed colony cells to irradiated CBA mice produced low numbers of spleen colonies, mainly erythroid in composition. Karyotypic analysis using the T6T6 marker chromosome showed that some of these colonies were of donor origin. With an assumed f factor of 0.2, the mean content of spleen colony-forming cells per 7-day mixed colony was calculated to vary from 0.09 to 0.76 according to the type of mixed colony assayed. The fetal and adult multipotential hemopoietic cells forming mixed colonies in agar may be hemopoietic stem cells perhaps of a special or fetal type.  相似文献   

3.
Interleukin-4 (IL-4), which was originally identified as a B-cell growth factor, has been shown to produce diverse effects on hemopoietic progenitors. The present study investigated the effects of purified recombinant murine IL-4 on early hemopoetic progenitors in methylcellulose culture. IL-4 supported the formation of blast cell colonies and small granulocyte/macrophage (GM) colonies in cultures of marrow and spleen cells of normal mice as well as spleen cells of mice treated with 150 mg/kg 5-fluorouracil (5-FU) 4 days earlier. When the blast cell colonies were individually picked and replated in cultures containing WEHI-3 conditioned medium and erythropoietin (Ep), a variety of colonies were seen, including mixed erythroid colonies, indicating the multipotent nature of the blast cell colonies supported by IL-4. To test whether or not IL-4 affects multipotent progenitors directly, we replated pooled blast cells in cultures under varying conditions. In the presence of Ep, both IL-3 and IL-4 supported a similar number of granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies. However, the number of GM colonies supported by IL-4 was significantly smaller than that supported by IL-3. When colony-supporting abilities of IL-4 and IL-3 were compared using day-4 post-5-FU spleen and day-2 post-5-FU marrow cells, IL-4 supported the formation of fewer blast cell colonies than did IL-3. IL-4 and IL-6 revealed synergy in support of colony formation from day 2 post-5-FU marrow cells. These results indicate that murine IL-4 is another direct-acting multilineage colony-stimulating factor (multi-CSF), similar to IL-3, that acts on primitive hemopoietic progenitors.  相似文献   

4.
The effect of retinoic acid (RA) on the colony-stimulating-factor-dependent clonal growth of myeloid progenitors was assessed in semisolid agar cultures of mouse bone marrow cells using L-cell-conditioned medium that gave rise to macrophage colonies, granulocyte colonies, and mixed macrophage-granulocyte colonies and clusters. RA was found to enhance the overall formation of myeloid colonies (about 50%) and clusters in 7-day cultures. The increase was due to an enhanced formation of macrophage colonies (70-250%) and clusters which reached a maximal value at about 3 microM RA. In 4-day cultures, the effect of RA on macrophage colony formation was biphasic with a maximal enhancement at 10 nM. RA suppressed granulocyte-colony formation in 4-day cultures. RA increased the phagocytic activity of bone-marrow-derived macrophages at all stages of differentiation and/or maturation in culture. The Fc-receptor-mediated erythrophagocytosis as well as the phagocytosis of heat-killed yeast cells (HK-yeast) and starch particles increased by RA treatment in a dose-dependent manner, reaching an increase of 100-200% of the activity expressed in the absence of RA. Peritoneal exudate macrophages likewise exhibited an increased phagocytic response to a variety of particles, at both physiological and pharmacological concentrations of RA. Expression of an RA-mediated increase in phagocytic activity required a prolonged incubation with RA (greater than 19 hr). The data suggest that RA may be of physiological relevance in the regulation of proliferation and function of hemopoietic cells. Therapeutic doses of RA may potentiate macrophage proliferation and function, elements that are crucial at all phases of the various defense mechanisms that the organism possesses.  相似文献   

5.
We studied the effects of interleukin-3 (IL-3) on colony formation by hemopoietic progenitors in methylcellulose cultures of spleen cells from 5-fluorouracil (FU)-treated mice. Purified IL-3 supported the growth of various types of multilineage colonies including blast cell colonies. The types of colonies were similar to those supported by pokeweed-mitogen spleen cell conditioned medium (PWM-SCM), except that IL-3 supported eosinophil and neutrophil expression better. Delayed addition of IL-3 to cultures 7 days after cell plating decreased the number of colonies to one-half the number in cultures with IL-3 added on day 0. It did not alter the proliferative and differentiation characteristics of late emerging multipotential blast cell colonies. These observations suggest that IL-3 does not trigger hemopoietic progenitors into active cell proliferation but is necessary for their continued proliferation. This permissive role of IL-3 is consistent with a stochastic model of stem cell proliferation which features random entry into cell cycle. IL-3 also supported the growth of multilineage colonies from single cells isolated from blast cell colonies by micromanipulation. This result shows that IL-3 acts directly on multipotential progenitors. Analysis of colonies derived from paired progenitors revealed disparate lineage expression and was in accordance with the stochastic model of stem cell differentiation.  相似文献   

6.
Tracing the progeny of the aortic hemangioblast in the avian embryo   总被引:9,自引:0,他引:9  
A population of hematopoietic progenitors becomes committed within the embryo proper in the floor of the aorta (P-Sp/AGM in the mouse). In birds, this first aspect of intraembryonic hematopoiesis is prominent during embryonic day 3 (E3) as endothelium-associated "intra-aortic clusters." Between E6 and E8, diffuse hematopoiesis then occurs as "para-aortic foci" located in the dorsal mesentery ventral to the aorta. These foci are not associated with endothelium. Whether these two hematopoietic cell populations arise from distinct or common progenitors is not known. We could recently trace back the origin of intra-aortic clusters in the avian embryo by labeling aortic endothelial cells (EC) in vivo with acetylated low-density lipoproteins. This approach established the derivation of early intraembryonic hemopoietic cells from the endothelium, but did not indicate how long during ontogeny such a relationship may exist, since the progeny of EC labeled at E2 could be traced for 1-2 days at most. Here we report that, when E2 aortic ECs were infected prior to the formation of intra-aortic clusters with a nonreplicative LacZ-bearing retroviral vector, numerous cells were labeled in the para-aortic foci at E6. In contrast, when the retroviral vector was inoculated at E4 rather than E2, that is, after the disappearance of intra-aortic clusters, no cells in the para-aortic foci were labeled. Taken together, our results demonstrate that ECs from the aortic floor seed the two aspects of aorta-associated hemopoiesis and that these ECs with hemangioblastic potential are present only transiently in the aorta.  相似文献   

7.
M Ogawa  S C Clark 《Blood cells》1988,14(2-3):329-337
Interleukin-6 (Il-6), also known as B cell stimulatory factor 2/interferon beta 2, has been found to support colony formation by murine granulocyte-macrophage progenitors. We have reported that Il-6 also acts synergistically with interleukin-3 (Il-3) in the support of the proliferation of multipotential stem cells in the quiescent, Go phase of the cell cycle. Our serial observations (mapping studies) of the development of blast cell colonies from spleen cells harvested from mice 4 days after the injection of 150 mg/kg 5-fluorouracil revealed that the blast cell colonies appeared earlier in culture in the presence of Il-6 and Il-3 than with either factor alone. Because the combination of factors did not alter the rate of growth of the colony, this effect must result from an early exit from Go. In the human system using purified, My-10+ bone marrow progenitors in a culture system with delayed addition of growth factors, the combination of Il-6 and Il-3 yielded twice as many colonies as did Il-3 alone. The human blast cell colonies also appeared at earlier times when grown in the presence of Il-6 and Il-3 as compared to either factor alone. These results suggested that human Il-6 acts synergistically with Il-3 in the support of the proliferation of human and murine hemopoietic stem cells in Go and that part of the effect appears to be a shortening of the Go residence time of the hemopoietic stem cells.  相似文献   

8.
J F Carmier  J Samarut 《Cell》1986,44(1):159-165
To determine the function of c-fps in chicken macrophages and granulocytic cells we have infected chicken bone marrow cells with retroviruses containing the v-fps oncogene. Normal chicken macrophage progenitors, M-CFCs, give rise to macrophage colonies in semisolid cultures when macrophage colony stimulating factor (M-CSF) is added into the culture medium. Upon infection with v-fps bearing retroviruses, we observed that M-CFCs were induced to develop macrophage colonies in vitro without exogenous M-CSF. This activation results from a direct effect of v-fps on the M-CFCs. No leukemic transformation was observed in the infected colonies. By comparing the effects of several retroviruses, we showed that the induction of M-CFC development is specific to v-fps containing viruses and mediated by the v-fps protein. These observations support the hypothesis that the c-fps gene is involved in the control of proliferation and/or differentiation of myeloid cells.  相似文献   

9.
We have previously demonstrated in quail embryos grafted on chick yolk sacs the existence of intraembryonic stem cells responsible for definitive hemopoiesis. In order to determine the origin of these cells, we now examine the diffuse hemopoietic processes within the avian embryo's mesoderm. At 4–5 days of incubation in the two species, basophilic cells were found throughout the dorsal mesentery. At 6–8 days these cells became very numerous and built up dense foci at the level of branching of the anterior and posterior cardinal veins. These cells often infiltrated the wall of lymph spaces and channels and were also present in the lumen of blood vessels. Such locations support the interpretation that these basophilic cells represent early stages of hemopoietic differentiation. At 8–10 days, erythropoiesis or granulopoiesis was seen in the foci, which then regressed rapidly. The foci maximal development coincided with the period of colonization of the intraembryonic organ rudiments. In “yolk sac chimeras,” the foci were always constituted by quail cells, indicating their intraembryonic origin. The primordial origin of the intramesodermal cells remains to be determined. A likely source might be the ventral wall of the aorta which appeared to shed cells into the lumen and into the mesentery in the 3-day embryo.  相似文献   

10.
The humoral influence of cells of hemopoietic organs of chicken embryos of different terms on the development of the colony and cluster formation of mononuclears of the bone marrow of mice was studied in joint cultivation in two-compartment cylindrical diffuse microchambers. The process of formation of colonies and clusters is inhibited by cells of the yolk sac on the 2nd-4th day of the development, by cells of the liver on the 8th-12th day, of the spleen on the 13th-18th day and of the bone marrow--on the 15th day. The yolk sac cells were found to have most considerable inhibiting influence on proliferation and differentiation of cells on the 2nd day of the development of chicken embryo. The yolk sac cells on the 6th day stimulate the formation of colonies and clusters. The yolk sac, beginning from the 4th day of the development, and the liver release humoral factors promoting the formation of erythroid colonies. The erythroid colonies are formed but when cultivated on the vascular membrane of the chicken embryo; the erythroid colonies are not formed when cultivated in the abdominal cavity of mice. Local erythropoietinoid factors are not synthetized by the spleen and bone marrow cells. A supposition is put forward that a combination of the local inhibiting and erythropoietic effects promotes the erythroid differentiation of cells.  相似文献   

11.
E-P. Ma  X-Z. Liu  M-D. Liu  Y. Han  X. Lui  Z-Z. Wu 《Cryobiology》1998,37(4):372-375
The effect of superoxide dismutase (SOD) on the recovery of human hemopoietic stem cells stored at 4°C has been studied. Human bone marrow cells from cancer patients without hemopoietic diseases were stored at 4°C for 1–7 days in RPMI 1640 medium containing 10% human AB serum with or without 6 U/ml of SOD. The recovery of colony-forming units of the following types were counted: granulocyte and macrophage type; megakaryocyte type; mixed granulocyte, erythrocyte, megakaryocyte, and macrophage type; and burst-forming units of erythrocytic type. The SOD groups recovered, respectively, 5.6, 5.3, 4.6, and 4.4 times as many colonies as the control group. This study demonstrates that the addition of SOD to hypothermic storage media may increase the recovery of hemopoietic stem cells.  相似文献   

12.
We recently identified a murine hemopoietic stem cell colony which consists of undifferentiated (blast) cells and appears to be more primitive than CFU-GEMM in the stem cell hierarchy. The progenitors for the colony which we termed “stem cell colony” possess an extensive self-renewal capacity and the ability to generate many secondary multipotential hemopoietic colonies in culture. We replated a total of 68 stem cell colonies from cultures of murine spleen cells and analyzed the number of stem cell–and granulocyte(neutrophil)-erythrocyte-macrophage-megakaryocyte (GEMM) colony-forming cells in individual stem cell colonies. Of the 68 stem cell colonies, 35 contained progenitors (abbreviated as “S”-cells) for stem cell colonies. The distributions of S-cells and CFU-GEMM in individual stem cell colonies were extremely heterogeneous. Neither the frequency distributions of S-cells nor CFU-GEMM in stem cell colonies could be fitted well by Poisson distribution. Rather, the frequency distribution of the s-cells could be approximated by a geometric distribution and that of CFU-GEMM by an exponential distribution, both of which are variates of the gamma distribution. Our observations are in agreement with those on the distributions of CFU-S in individual spleen colonies and provided support for a stochastic model for stem cell self-renewal and commitment in culture. Application of the theory of the branching process to the distribution of S-cells revealed a distributional parameter “p” of 0.589 which is also in agreement with the earlier report on the p value for reproduction of CFU-S.  相似文献   

13.
The integrin chain alphaV, expressed in association with beta3, by cells of the megakaryocytic/thrombocytic and endothelial lineages is thought to play an important role in angiogenesis. alphaVbeta3 expression by endothelial cells is not constitutive but induced by various stimuli in avian and human models. Here the developmental pattern of alphaVbeta3 expression was analysed in the chick embryo by immunocytochemistry, using a specific monoclonal antibody. On day 2 of development alphaVbeta3 expression was restricted to rare cells in the blood stream, in the embryo proper and in the yolk sac blood islands. AlphaVbeta3 expression by endothelial cells became detectable on day 3 and was restricted to the dorsal aorta. Interestingly it was absent from the intra-aortic hemopoietic clusters (E3.5) which, as we have showed previously, express the alphaIIbbeta3 integrin and display progenitor potentialities. However the endothelium underlying intra-embryonic hemopoietic clusters expressed this integrin. In contrast E6-7 para-aortic hemopoietic foci contained numerous alphaVbeta3 positive cells. Both alphaVbeta3 and alphaIIbbeta3 were expressed in these latter hemopoietic sites, while alphaVbeta3 was still selectively expressed by the aortic endothelium until E6. Thereafter, at E7 the pulmonary artery also expressed it. Since alphaIIbbeta3 is expressed by avian and murine multilineage hemopoietic progenitors, we then studied the hemopoietic potentialities of alphaVbeta3/alphaIIbeta3 double positive cells from embryonic bone marrow differentiating in vitro in erythro-myeloid conditions. Thrombocytic, erythroid and myeloid progenitor potentialities were found within the cell population expressing both beta3 integrins.  相似文献   

14.
The ability of yolk sac and primary bone marrow cells of the quail to form hemopoietic colonies at 6 hours of incubation (i. e. before establishment of circulation) was studied in the bone marrow of 3-week sublethally irradiated chickens. The experiments were based on the possibility of differentiating between quail and chicken cells from the natural cell marker (Pheulgen-positive nucleolus). The number of hemopoietic colonies produced by cells transplanted from the primary bone marrow was three times greater than that consequent on transplantation of yolk sac cells. With the given dose of irradiation the bone marrow shows about 75% exogenous (quail) and 25% endogenous (chicken) hemopoietic colonies.  相似文献   

15.
The cells responsible for the long-term in vitro generation of murine mast cells have been examined. Sequential analysis of all colony types obtained from cultures of spleen or bone marrow cells showed that only colonies derived from multipotential cells (mixed-erythroid colonies) or mast cell progenitors, contained cells responsible for mast cell generation in liquid cultures. Primary colony growth and subsequent maintenance of mast cells in liquid cultures was dependent upon pokeweed mitogen-stimulated spleen cell-conditioned medium (SCM). Mixed-erythroid colonies from 14-day cultures of spleen cells had the greatest capacity for mast cell generation. Analysis by clone splitting and transfer to high (20%) and low (2.5%) concentrations of SCM showed that the concentration of SCM used in either the primary colony culture or subsequent liquid culture phase altered both the proliferative capacity of the mast cells generated and the frequency of mast cell progenitors within individual mixed-erythroid colonies. Thus, mixed-erythroid colonies stimulated with 2.5% SCM contained the highest proportion of mast cell progenitors (34% of colonies) and when stimulated with 20% SCM, approximately fourfold higher numbers of mast cells were produced at weekly intervals from liquid cultures maintained in 2.5% SCM compared to parallel liquid cultures containing 20% SCM. These studies confirm the hemopoietic origin of mast cells and demonstrate that a factor(s) in SCM is able to modulate their proliferative potential.  相似文献   

16.
Photodynamic therapy represents a new approach for the local control of cancers. It has recently been claimed that photodynamic therapy mediated by hematoporphyrin derivative (HPD) is selectively more efficient for killing leukemic cells than normal progenitors. To improve this effect, we studied the influence of hematoporphyrin dose, temperature during incubation and/or treatment, hematoporphyrin derivative incubation time, and fractionation of the argon laser light (488-514 nm) used for hematoporphyrin stimulation. Plating efficiency calculated after a 7-day period of growth on collagen gel medium showed a dose-dependent phototoxicity of HPD reaching 0.01% for normal hemopoietic progenitors and 0.001% for leukemic cells (dose = 12.5 micrograms/ml). The 10:1 ratio of normal hemopoietic progenitors to leukemic cells was also found to be the same or increased when temperature was 37 degrees C during incubation and 4 degrees C during laser irradiation. Similar results were also found when incubation time was varied from 75-120 min, or when laser irradiation dose was fractionated into 2 or 3 periods. The ratio of normal progenitors to leukemic cells reached 100:1 when 75 J/cm2 were fractionated into 3 periods after an incubation time of 120 min with 10 micrograms/ml HPD. Selectivity in photodynamic treatment seems to occur between normal hemopoietic progenitors and leukemic cells. The mechanism of this selectivity remains unclear, but experiments with the fractionated irradiation dose suggest that as in radiotherapy, better potentially lethal damage repair in normal cells could be a factor for selectivity in photodynamic therapy. Our results obtained with leukemic cells are fully in agreement with data in the literature concerning similar experimental models.  相似文献   

17.
18.
Cell sorting has been used as a method for characterizing hemopoietic stem cells and progenitors. Fluorescent antibody-surface labels and changes in fluorescence polarization induced by in vitro stimulation with potential hemopoietic regulators were used. As detected by significant enrichment of CFU-S (pluripotent stem cells) in fluorescence-activated cell sorting, some CFU-S bear 'unique antigens' recognized by rabbit anti-human brain sera, human anti-human sperm sera, and 129 anti-F9 serum, but not A . TH anti-A . TL (Ia) ascites. Significant changes in fluorescence polarization induced by in vitro stimulation of mouse bone marrow with potential hemopoietic regulators were also observed; further, progenitors of human T-lymphocyte colonies were observed to exhibit a significantly decreased mean polarization value after short-term stimulation with PHA-LCM (phytohemagglutinin-stimulated leukocyte conditioned medium).  相似文献   

19.
We have investigated the developmental relationship of the hemopoietic and endothelial lineages in the floor of the chicken aorta, a site of hemopoietic progenitor emergence in the embryo proper. We show that, prior to the onset of hemopoiesis, the aortic endothelium uniformly expresses the endothelium-specific membrane receptor VEGF-R2. The onset of hemopoiesis can be precisely determined by detecting the common leukocyte antigen CD45. VEGF-R2 and CD45 are expressed in complementary fashion, namely the hemopoietic clusters in the floor of the aorta are CD45+/VEGF-R2-, while the rest of the aortic endothelium is CD45-/VEGF-R2+. To determine if the hemopoietic clusters are derived from EC, we tagged the E2 endothelial tree with a non-replicative retroviral vector and low density lipoproteins. Twenty four-48 hours later, labelled cells in the vascular tree were found to be either endothelial or hemopoietic but exceptionally both. Another 1-2 days later, groups of labelled cells appear in the dorsal mesentery within the hemopoietic "paraortic foci". Since no CD45+ cells were inserted among endothelial cells at the time of vascular labelling, hemopoietic clusters and foci must be concluded to derive from precursors with an endothelial phenotype.  相似文献   

20.
The yolk sac of the 12-day chicken embryo retains the blast stage progenitors to cells of the myeloid lineages with a very low level of contamination by more mature myeloid cells which have begun to express the characteristic myeloid cell markers. Both in vivo and in vitro experiments have supported the hypothesis that target cells for the BAI-A strain of avian myeloblastosis virus are contained within the myeloid lineages. An assay system for avian myeloblastosis virus was developed which utilizes this yolk sac cell system and which appears to be more sensitive than previous published assays. In addition, the kinetics of a liquid culture transformation system is presented in which at least 4% of the yolk sac cell population was transformed in a relatively synchronous fashion at 2 days after infection. The morphological transformation preceded an increased rate of cell proliferation. Cell separation procedures provided a 10- to 20-fold enrichment of target cells and demonstrated that the target cell population copurifies with macrophage colony-forming cells which are the committed progenitors to the macrophage lineage. In combination with earlier work, this work demonstrated that cells committed to the macrophage lineage at all stages of differentiation may serve as target cells for infection by avian myeloblastosis virus.  相似文献   

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