Regulation of Hemopoietic Stem Cell Turnover and Population Size in Neonatal Mice (original) (raw)
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The effect of stem cell proliferation regulators demonstrated with an in vitro assay
Blood, 1988
Spleen colony formation after transplantation of bone marrow cells into irradiated mice has been used as an assay for hematopoietic stem cells (CFU-S), but has serious limitations intrinsic to an in vivo assay. In this report we describe experiments using an in vitro clonogenic assay that is especially suitable for studies of stem cell regulation as defined growth factors and normal untreated bone marrow can be used. We have demonstrated that the colony-forming cells have proliferative properties in common with CFU-S and respond to specific proliferation regulators previously detected using the spleen colony assay.
Aspects of the biology of the neonatal hematopoietic stem cell
STEM CELLS, 1996
We have studied the frequency of colony forming cells (CFC) in fetal and neonatal blood in comparison with adult blood and marrow. Fetalheonatal blood contains at least as many CFC as adult marrow and higher numbers of the more primitive CFC-those CFC giving rise to colonies composed of erythroid and myeloid cells. CD34' cord blood cells (selected either by sorting, panning or affinity chromatography) proliferate in culture over time and generate more CFC (from pre-CFC) and differentiated cells in response to Steel factor plus different hematopoietic growth factors. Steel factor is unable to stimulate cell growth by itself under serum-deprived conditions and requires the synergistic action of erythropoietin (Epo), granulocyte colony stimulating factor (G-CSF) or interleukin 3 (IL-3). In the presence of Epo or G-CSF, CFC and differentiated cells are generated for 15 days and are mainly erythroid or granulocytic, respectively. In contrast, Steel factor plus IL-3 generates multilineage CFC and differentiated cells for more than one month. When the conditions for these long-term suspension cultures were optimized (37"C, regular refeeding with fresh growth factors and media without changing the flask), CFC and differentiated cells were generated for more than two months. At this time, CFC were no longer detectable and all cells had a mast cell phenotype. These cells have been maintained and propagated for more than eight months in the presence of IL-3 and Steel factor and may represent a useful tool to study human mast cell differentiation. Finally, the addition of oligonucleotides antisense to c-kit, the receptor for Steel factor, selectively suppresses the generation of erythroid . OAlphaMed Press 1066-5099/93/$5.00/0 cells, indicating that Steel factorlc-kit interaction plays a major role in the process of erythroid commitment.
Study on the proliferative state of haemopoietic stem cells (CFU)
1976
Hydroxyurea was used to study the proliferation rate of haemopoietic stem cells (CFU3 in normal mice, after irradiation or transplantation into irradiated recipients. It was demonstrated that the proliferation rate of endogenous CFU, (endo-CFU,) and exogenous CFU, (exo-CFUs) are identical. After irradiation (650 R) the surviving endo-CFU, begin to proliferate immediately. By contrast exo-CFU, transplanted into the irradiated recipient mouse (850 R), begin to proliferate onty after about 30 hr. However, injection of isoproterenol (which stimulates adenyl cyclase) or dibutyryl cyclic adenosine 3',5'-monophosphate shortly after marrow cell graft, triggers the transplanted CFU, into cell cycle as shown by an almost immediately increased sensitivity to hydroxyurea. Isoproterenol is capable of inducing DNA synthesis also in stem cells of normal mice but it takes about 20 hr before CFU, become to be increasingly sensitive to hydroxyurea. Multipotential haemopoietic stem cells (CFU,) can be quantitatively measured in mice by methods developed by Till & McCulloch (1961). One method is based on transplantation of small amount of bone marrow or spleen cells into the supralethally irradiated recipients and counting colonies on the spleen 8-10 days later (exo-CFU,). The other method follows the development of colonies in spleen from the stem cells which had survived sublethal irradiation of the animals (endo-CFU,). The pioneering studies of Becker, McCulloch & Till (1965), Lajtha et af. (1969) and others demonstrated that CFU, represent a slowly proliferating population with approximately only 10 % of cells in the S phase of the cell cycle. Recently Boggs et al. (1973) and Boggs & Boggs (1973) suggested that endo-CFU, have a higher proliferative rate as compared to exo-CFU,. Hydroxyurea, the inhibitor of ribonucleotide reductase (Skoog & Nordenskjtild, 1971) was shown to be suitable for in uiuo determination of the fraction of cells in the S phase (Morse, Rencricca & Stohlman, 1970; Kubanek et af., 1973). We have re-investigated the proliferative state of exo-and endo-CFU, by means of hydroxyurea.
Expansion and Differentiation of Immature Mouse and Human Hematopoietic Progenitors
Developmental Hematopoiesis, 2004
A prerequisite for proper investigation of self-renewal and differentiation of hematopoietic cells is the possibility to obtain large quantities of homogenous primary progenitors under defined conditions, allowing meaningful biochemical and molecular analyses. These cells should show renewal and differentiation characteristics similar to the in vivo situation. The serum-free culture systems delineated in this chapter meet these requirements, employing primary hematopoietic cells derived from murine fetal liver and human umbilical cord blood, which show physiological self-renewal responses to cytokine/hormone combinations, which in vivo are involved in stress hematopoiesis. We describe the expansion and sustained proliferation of multipotent (mouse) and erythroid (mouse and human) progenitors, responding to physiological signals. Moreover, both mouse and human erythroid progenitors can be induced to undergo synchronous terminal differentiation by addition of high levels of erythropoietin. If fetal liver cells from p53 -/mice are used, respective multipotent and erythroid cells undergo immortalization without an obvious Hayflick crisis, but otherwise retain their primary cell characteristics. Finally, both primary and immortal mouse progenitors can be subjected to genetic manipulation via retroviral constructs with high efficiency.
[Biology of hematopoietic stem cells]
1999
All the cells comprising the hemopoietic system are derived from a common precursor, the totipotent hemopoietic cell (THC), which, through processes of proliferation and differentiation, gives rise to all the mature cells found in the blood and lympho-hemopoietic organs. In order that the processes of proliferation, survival, apoptosis, and differentiation from THCs to mature cells take place, the participation of proteins denoted collectively as cytokines is required. Their role is to promote and regulate one or several functions (depending on the cell type and stage of development), and to participate in one or several stages of cell development of the THCs. By the use of different tissue culture techniques, it was concluded that other non-hemopoietic cell types have an important role. These cells are those comprising the stroma in the bone marrow: fibroblasts, endothelial cells and adipocytes among others. The contribution of the stroma lies in the production of cytokines, as wel...
Biological Procedures Online, 2007
In adults, hematopoiesis occurs in bone marrow (BM) through a complex process with differentiation of hematopoietic stem cells (HSCs) to immune and blood cells. Human HSCs and their progenitors express CD34. Methods on hematopoietic regulation are presented to show the effects of the chemokine, stromal-derived growth factor (SDF)-1α and the neuropeptide, substance P (SP). SDF-1α production in BM stroma causes interactions with HSCs, thereby retaining the HSCs in regions close to the endosteum, at low oxygen. Small changes in SDF-1α levels stimulate HSC functions through direct and indirect mechanisms. The indirect method occurs by SP production, which stimulates CD34 + cells, supported by ligand-binding studies, long-term culture-initiating cell assays for HSC functions, and clonogenic assays for myeloid progenitors. These methods can be applied to study other hematopoietic regulators.
Stem cell factor induces proliferation and differentiation of fetal progenitor cells in the mouse
British Journal of Haematology, 1998
Recombinant rat stem cell factor (SCF) was studied for its ability to stimulate the growth of murine hematopoietic progenitor cells and to generate colony-forming cells (CFC) from highly enriched populations of hematopoietic cells. In serum-deprived cultures, SCF alone stimulated few colonies but interacted with a number of other hematopoietic growth factors, particularly interleukin 3, to promote colony formation. The most marked effect was on the generation of mixed-cell colonies. Hematopoietic cells were sorted into wheat-germ agglutinin-negative, monocyte-depleted, rhodamine 123 (Rh123)-bright or Rh23-dull cells. Historically, Rhl23-bright cells are capable of short-term (<1 mo) marrow engraftment, whereas among Rh23-dull cells are cells capable of long-term marrow engraftment. Enriched cells (2.5 x 103) were placed into serum-deprived liquid cultures with various hematopoietic growth factors. Initially, the Rh123-bright and Rh123-dull cells had few CFC but, in the presence of interleukin 3 and SCF, Rh123-bright cells gave rise to >15,000 granulocyte/macrophage CFC, >1500 erythroid burstforming cells, and >700 mixed-cell CFC by day 5. In contrast, RhI23-dull cells proliferated only in the presence of interleukin 3 and SCF, but total cell numbers rose to a peak of 18,000 by day 21, and one-third of the cells were CFC. Thus, SCF, in combination with other growth factors, can generate large numbers of CFC from pre-CFC and appears to act earlier than hematopoietic growth factors described to date.
Immunity, 1996
Generally, these same cell surface markers also define fetal liver LTR-HSCs (Zeigler et al., 1994; Jordan et al., At day 10 in mouse gestation, the intraembryonic 1995). However, some phenotypic features unique to aorta-gonads-mesonephros (AGM) region generates fetal liver LTR-HSCs have been revealed: first, 13 and the first definitive hematopoietic stem cells (HSCs) of 14 days postcoitum (dpc) fetal liver cells are Mac-1 ϩ the adult blood system. By 11 days postcoitum, the (Morrison et al., 1995), whereas bone marrow LTR-HSCs liver contains such HSCs. While HSCs of the adult are Mac-1 Ϫ ; second, antigens AA4.1 (Jordan et al., 1990; bone marrow and late-stage fetal liver have been ex-Jordan and Lemischka, 1990) and CD45RB (Rebel et al., tensively characterized for cell surface markers, there 1996) are expressed in fetal liver population enriched has been no phenotypic description of the first HSCs for HSCs, whereas HSC activity is reported in both during embryo development. We report here the tem-AA4.1 ϩ and AA4.1 Ϫ adult bone marrow populations (Szilporal cell surface phenotype of HSCs from the AGM vassy and Cory, 1993; Orlic at al., 1993; Trevisan and region and early fetal liver and show that all HSCs Iscove, 1995; Rebel et al., 1996). reside in the c-kit ؉ population. c-kit ؉ HSCs from AGM The predominant hematopoietic organs in the preand liver are mainly CD34 ؉ and in the AGM are in both liver hematopoietic stage of mouse development (begin-Mac-1 ؉ and Mac-1 Ϫ fractions. These results demonning at 7.5 dpc) are the embryonic yolk sac (YS) (Russel strate that during mouse ontogeny the first definitive and Bernstein, 1966; Moore and Metcalf, 1970), para-HSCs are similar in cell surface phenotype to the HSCs aortic splanchnopleura (Godin et al., 1993; Dieterlenof adult bone marrow but that spatial localization and Liè vre and Le Douarin, 1993) and the aorta-gonadsdevelopmental time are critical factors in the phenomesonephros (AGM) region (Medvinsky et al., 1993; typic assessment of this functional cell population. Medvinsky, 1993; Mü ller et al., 1994; Dzierzak and Medvinsky, 1995). From 8-9 dpc, the extraembryonic YS as well as the intraembryonic para-aortic splanchnopleura