Growth kinetics of hemopoietic fetal stem cells

Fetal spleen stem cells have growth characteristics similar to those of normal adult spleen stem cells. On the contrary there is an early fetal liver stem cell population which possesses a lag time longer than that of adult stem cells. The duration of the lag time is controlled by a built-in biological timer which seems to regulate some proliferative functions of the primitive liver stem cell.     

The self renewal probability of hemopoietic stem cells

The probability of a colony which originated as a single stem cell to become extinct due to differentiation of all of its stem cells in any generation is closely connected to stem cell self renewal probability p. p can be determined from the coefficient of variation of the colony numbers received by reinjecting single colonies of the same age. Whole spleens containing a known average colony number can also be used with advantage for this purpose. The results of both procedures indicate a stem cell self renewal probability p =0.62 ± 0.04, which does not change significantly between the sixth and the fourteenth day of colony development, and an extinction probability ω = 0.63 ± 0.12.     

Pluripotent hemopoietic stem cells give rise to osteoclasts in vitro: effects of rGM-CSF

Studies involving bone marrow transplantation of osteopetrotic rodents have provided evidence for the lineage of the ostcoclast. Recent investigations have demonstrated that pluripotent hemopoietic stem cells (PHSC) isolated from the bone marrow of normal animals cure the skeletal sclerosis and result in the formation of normal osteoclasts when transplanted into ia osteopetrotic rats. A criticism of these findings is that the microenvironment of the osteopetrotic bone and the bone marrow compartment may be unique in its ability to induce the differentiation of these stem cells into osteoclasts. To test this hypothesis, PHSC were co-cultured with fetal metatarsal bones from normal animals. PHSC were isolated from normal bone marrow using FITC-labelled monoclonal antibodies directed against rat Thy 1.1 and fluorescence-activated cell sorting. The PHSC or whole mononuclear bone marrow were co-cultured with 20-day fetal rat metatarsal rudiments. In some cultures, recombinant mouse granulocyte-macrophage colony-stimulating factor (rGM-CSF) (250 U per culture) was added in addition to the PHSC. After 7 days the fetal bones were prepared for light and electron microscopy and the number of osteoclasts generated in vitro was determined. The PHSC isolate generated as many osteoclasts as the whole mononuclear bone marrow. The addition of rGM-CSF did not enhance the generation of osteoclasts in either control bones or in bones cultured with PHSC. These results are equivalent to those reported in the osteopetrotic transplant system.     

The effect of carnosine on hematopoietic stem cell activity in irradiated animals]

Carnosine given to adult animals together with potable water one day prior to gamma-irradiation or injected in a single intraperitoneal dose one hour after irradiation enhances colony formation by haemopoietic stem cells migrating from the bone marrow to the spleen. In young animals with a high colony-forming activity carnosine either decreases or does not influence at all the efficiency of colony production.     

Pluripotent hemopoietic stem cells in mice and humans

Although it has been reported previously that pluripotent hemopoietic stem cells (P-HSCs) express c-kit, the receptor for stem cell factor (steel factor), we and other groups have recently shown that P-HSCs do not express c-kit. In this review, we provide evidence that c-kit 2 years) and the capacity to form colony-forming units in spleen (CFU-S) on Day 16, although c-kit(low) HSCs or c-kit+ HSCs have LTRA less than 1.5 years and the capacity to form CFU-S on Day 14 or on Day 10, respectively. In addition, we have found that there is a major histocompatibility complex (MHC) restriction between P-HSCs and stromal cells; normal P-HSCs can proliferate and differentiate efficiently in collaboration with MHC class I-compatible (but not MHC class I-incompatible) stromal cells. In humans, we also show that c-kit相似文献   

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

The dilution factor of intravenously injected hemopoietic stem cells

In order to measure the total number of stem cells present in a given preparation, it is necessary to know the fraction of stem cells which settle in the spleen and form macroscopically visible colonies. The intravenous injection does not allow a direct measurement of this fraction. However, its value is established by injecting stem cells directly into the spleen, thus minimizing the dilution of stem cells through the circulation. It has been measured with this procedure, that only 4% of intravenously injected stem cells form spleen colonies.     

The renewal and differentiation of hemopoietic stem cells.

Blood-forming tissues are organized in well-defined microenvironments composed of hemopoietic cells and a supportive stroma of connective tissue and endothelium. Hemopoietic cells segregate to various lineages, all derived from a small population of pluripotent stem cells residing in the bone marrow. Regulation of growth and differentiation, particularly under conditions of perturbations, damage, and disease, is mediated by inducer colony-stimulating factors and interleukins counteracted by inhibitory cytokines. Whereas much is known about the mode of induction of differentiation, insufficient information is available to explain the process of stem cell renewal that is crucial for the longevity of the hemopoietic system. It is also only partially known how inhibition of hemopoietic processes occurs, and what molecules in blood-forming tissues signal organization into discrete patterns. This paper reviews recent progress that has opened new avenues to a better understanding of this highly complex issue.     

In vitro production of hemopoietic stem cells: washings from organ cultures of embryonic liver]

Hemopoietic cells including CFUs could be washed off from the organ culture of fetal liver periodically for 4 weeks. Under the cultivation conditions employed this treatment did not reduce the CFUs content of the culture essentially; thus, the washings off could be used to elevate the CFUs yield per culture.     

Gene transfer into hemopoietic stem cells using retroviral vectors

Gene transfer to hemopoietic cells offers a variety of new approaches to the experimental hematologist as well as potentially providing a means for correcting a number of genetic disorders of humans. From the experimental viewpoint, gene transfer utilizing retroviral vectors introduces new methods for analyzing hemopoietic cell lineages, and the effects of over-expression of genes affecting hemopoietic cell proliferation and differentiation. The unique properties of retroviral vectors and the optimized methods currently in use to infect hemopoietic cells are represented as a brief review of a rapidly expanding new field of experimental hematology.     

Physical separation of hemopoietic stem cells from cells forming colonies in culture

Mouse bone marrow cells in suspension were separated into a number of fractions on the basis of cell density by equilibrium density gradient centrifugation, or on the basis of cell size by velocity sedimentation. After each type of separation, the cells from the various fractions were assayed for their ability to form macroscopic spleen colonies in irradiated recipient mice, and for their ability to form colonies in a cell culture system. The results from either separation technique demonstrate that cells in some fractions formed more colonies in vivo than in the culture system, while cells in other fractions formed more colonies in culture than in the spleen. The results of control experiments indicate that this separation of the two types of colony-forming cells was not an artifact of the separation procedures. From these experiments it was concluded that the population of cells which form colonies in culture under the conditions used is not identical to the population of cells detected by the spleen colony assay.     

IFN-gamma negatively modulates self-renewal of repopulating human hemopoietic stem cells

Whereas multiple growth-promoting cytokines have been demonstrated to be involved in regulation of the hemopoietic stem cell (HSC) pool, the potential role of negative regulators is less clear. However, IFN-gamma, if overexpressed, can mediate bone marrow suppression and has been directly implicated in a number of bone marrow failure syndromes, including graft-vs-host disease. Whether IFN-gamma might directly affect the function of repopulating HSCs has, however, not been investigated. In the present study, we used in vitro conditions promoting self-renewing divisions of human HSCs to investigate the effect of IFN-gamma on HSC maintenance and function. Although purified cord blood CD34(+)CD38(-) cells underwent cell divisions in the presence of IFN-gamma, cycling HSCs exposed to IFN-gamma in vitro were severely compromised in their ability to reconstitute long-term cultures in vitro and multilineage engraft NOD-SCID mice in vivo (>90% reduced activity in both HSC assays). In vitro studies suggested that IFN-gamma accelerated differentiation of targeted human stem and progenitor cells. These results demonstrate that IFN-gamma can negatively affect human HSC self-renewal.     

Stimulating cROSstalk between commensal bacteria and intestinal stem cells

EMBO J (2013) 32 23, 3017–3028 10.1038/emboj.2013.224; published online October182013Commensal gut bacteria benefit their host in many ways, for instance by aiding digestion and producing vitamins. In a new study in The EMBO Journal, Jones et al (2013) report that commensal bacteria can also promote intestinal epithelial renewal in both flies and mice. Interestingly, among commensals this effect is most specific to Lactobacilli, the friendly bacteria we use to produce cheese and yogurt. Lactobacilli stimulate NADPH oxidase (dNox/Nox1)-dependent ROS production by intestinal enterocytes and thereby activate intestinal stem cells.The human gut contains huge numbers of bacteria (∼10¹⁴/person) that play beneficial roles for our health, including digestion, building our immune system and competing with harmful microbes (Sommer and Backhed, 2013). Both commensal and pathogenic bacteria can elicit antimicrobial responses in the intestinal epithelium and also stimulate epithelial turnover (Buchon et al, 2013; Sommer and Backhed, 2013). In contrast to gut pathogens, relatively little is known about how commensal bacteria influence intestinal turnover. In a simple yet elegant study reported recently in The EMBO Journal, Jones et al (2013) show that among several different commensal bacteria tested, only Lactobacilli promoted much intestinal stem cell (ISC) proliferation, and it did so by stimulating reactive oxygen species (ROS) production. Interestingly, the specific effect of Lactobacilli was similar in both Drosophila and mice. In addition to distinguishing functional differences between species of commensals, this work suggests how the ingestion of Lactobacillus-containing probiotic supplements or food (e.g., yogurt) might support epithelial turnover and health.In both mammals and insects, ISCs give rise to intestinal enterocytes, which not only absorb nutrients from the diet but must also interact with the gut microbiota (Jiang and Edgar, 2012). The metazoan intestinal epithelium has developed conserved responses to enteric bacteria, for instance the expression of antimicrobial peptides (AMPs; Gallo and Hooper, 2012; Buchon et al, 2013), presumably to kill harmful bacteria while allowing symbiotic commensals to flourish. In addition to AMPs, intestinal epithelial cells use NADPH family oxidases to generate ROS that are used as microbicides (Lambeth and Neish, 2013). High ROS levels during enteric infections likely act non-discriminately against both commensals and pathogens, but controlled, low-level ROS can act as signalling molecules that regulate various cellular processes including proliferation (Lambeth and Neish, 2013). In flies, exposure to pathogenic Gram-negative bacteria has been reported to result in ROS (H₂O₂) production by an enzyme called dual oxidase (Duox; Ha et al, 2005). Duox activity in the fly intestine (and likely also the mammalian one) has recently been discovered to be stimulated by uracil secretion by pathogenic bacteria (Lee et al, 2013). In the mammalian intestine another enzyme, NADPH oxidase (Nox), has also been shown to produce ROS in the form of superoxide (O₂⁻), in this case in response to formylated bacterial peptides (Lambeth and Neish, 2013). A conserved role for Nox in the Drosophila intestinal epithelium had not until now been explored.Jones et al (2013) checked seven different commensal bacterial to see which would stimulate ROS production by the fly''s intestinal epithelium, and found that only one species, a Gram-positive Lactobacillus, could stimulate significant production of ROS in intestinal enterocytes. Five bacterial species were checked in mice or cultured intestinal cells, and again it was a Lactobacillus that generated the strongest ROS response. Although not all of the most prevalent enteric bacteria were assayed, those others that were—such as E. coli—induced only mild, barely detectable levels of ROS in enterocytes. Surprisingly, although bacteria pathogenic to Drosophila, like Erwinia caratovora, were expected to stimulate ROS production via Duox, Jones et al (2013) did not observe this using the ROS detecting dye hydrocyanine-Cy3, or a ROS-sensitive transgene reporter, Glutatione S-transferase-GFP, in flies. Further, Jones et al (2013) found that genetically suppressing Nox in either Drosophila or mice decreased ROS production after Lactobacillus ingestion. Consistent with the important role of Nox, Duox appeared not to be required for ROS production after Lactobacillus ingestion. In addition, Jones et al (2013) found that Lactobacilli also promoted DNA replication—a metric of cell proliferation and epithelial renewal—in the fly''s intestine, and that this was also ROS- and Nox-dependent. Again, the same relationship was found in the mouse small intestine. Together, these results suggest a conserved mechanism by which Lactobacilli can stimulate Nox-dependent ROS production in intestinal enterocytes and thereby promote ISC proliferation and enhance gut epithelial renewal.In the fly midgut, uracil produced by pathogenic bacteria can stimulate Duox-dependent ROS production, which is thought to act as a microbicide (Lee et al, 2013), and can also promote ISC proliferation (Buchon et al, 2009). However, Duox-produced ROS may also damage the intestinal epithelium itself and thereby promote epithelial regeneration indirectly through stress responses. In this disease scenario, ROS appears to be sensed by the stress-activated Jun N-terminal Kinase (JNK; Figure 1A), which can induce pro-proliferative cytokines of the Leptin/IL-6 family (Unpaireds, Upd1–3) (Buchon et al, 2009; Jiang et al, 2009). These cytokines activate JAK/STAT signalling in the ISCs, promoting their growth and proliferation, and accelerating regenerative repair of the gut epithelium (Buchon et al, 2009; Jiang et al, 2009). It is also possible, however, that low-level ROS, or specific types of ROS (e.g., H₂O₂) might induce ISC proliferation directly by acting as a signal between enterocytes and ISCs. Since commensal Lactobacillus stimulates ROS production via Nox rather than Duox, this might be a case in which a non-damaging ROS signal promotes intestinal epithelial renewal without stress signalling or a microbicidal effect (Figure 1B). However, Jones et al (2013) stopped short of ruling out a role for oxidative damage, cell death or stress signalling in the intestinal epithelium following colonization by Lactobacilli, and so these parameters must be checked in future studies. Perhaps even the friendliest symbiotes cause a bit of ‘healthy'' damage to the gut lining, stimulating it to refresh and renew. Whether damage-dependent or not, the stimulation of Drosophila ISC proliferation by commensals and pathogens alike appears to involve the same cytokine (Upd3; Buchon et al, 2009), and so some of the differences between truly pathogenic and ‘friendly'' gut microbes might be ascribed more to matters of degree than qualitative distinctions. Future studies exploring exactly how different types of ROS signals stimulate JNK activity, gut cytokine expression and epithelial renewal should be able to sort this out, and perhaps help us learn how to better manage the ecosystems in our own bellies. From the lovely examples reported by Jones et al (2013), an experimental back-and-forth between the Drosophila and mouse intestine seems an informative way to go.Open in a separate windowFigure 1Metazoan intestinal epithelial responses to commensal and pathogenic bacteria. (A) High reactive oxygen species (ROS) levels generated by dual oxidase (Duox) in response to uracil secretion by pathogenic bacteria. (B) Low ROS levels generated by NADPH oxidase (Nox) in response to commensal bacteria. In addition to acting as a microbiocide, ROS in flies may stimulate JNK signaling and cytokine (Upd 1–3) expression in enterocytes, thereby stimulating ISC proliferation and epithelial turnover or regeneration. Whether this stimulation required damage to or loss of enterocytes has yet to be explored.