Behavior Of Hematopoietic Stem Cells Research Articles

Nitric Oxide Synthases Modulate Progenitor and Resident Endothelial Cell Behavior in Galactosemia

We used knockout animals of either inducible nitric oxide synthase (iNOS(/)) or endothelial NOS (eNOS(/)) to characterize the role of NOS in galactosemia, a model of diabetic retinopathy. NADH oxidase and nitrotyrosine were used as biomarkers of oxidative stress and vascular dysfunction. These animals were engrafted with hematopoietic stem cells (HSC) expressing green fluorescence protein (gfp(+)) to characterize the contribution of HSC and endothelial progenitor cells to neovascularization. Increased NADH oxidase activity and superoxide generation occurred in all galactose-fed mice. eNOS(/) mice demonstrated increased iNOS immunoreactivity in their retinal vasculature. Nitrotyrosine levels were low at baseline in the wild-type (WT) mice, eNOS(/) and iNOS(/) mice, and the galactose-fed iNOS mice and increased following galactose feeding in eNOS(/) and WT. Galactose-fed WT.gfp and iNOS(/).gfp chimeric animals had areas of perfused new vessels composed of gfp(+) cells. In contrast, galactose-fed eNOS(/).gfp mice produced copious, unbranched, nonperfused tubes. Thus, nitric oxide modulates HSC behavior and vascular phenotype in the retina. Although there is increased NADH oxidase and superoxide in galactosemic mice of all isoforms, iNOS is the source of nitric oxide responsible for peroxynitrite and nitrotyrosine formation that leads to the pathology observed in galactosemic mice.

Hematopoietic Stem Cells Can Give Rise to Satellite-Like Cells in Skeletal Muscles.

Recent studies have indicated that bone marrow cells can regenerate damaged muscles, but also that they can adopt phenotype of other cells by cell fusion. It has also been reported that single hematopoietic stem cells (HSCs) can regenerate skeletal muscle although it is still controversial whether HSCs differentiate into satellite cells in muscle or not. In order to investigate the roles of HSCs in muscle regeneration and whether they can generate satellite cells or not, we purified and injected CD45+Lin−Sca-1+c-kit+(CD45+KSL) HSCs labeled by green fluorescent protein (GFP) into mice with or without irradiation. We examined time-course behavior of HSCs in recipient muscles with a fluorescent stereomicroscope and then immunohitochemical staining during the early and late phase after transplantation. Our direct visualization system gave evidence of massive GFP signals in all the muscles of only irradiated mice in early phase after transplantation. Transverse cryostat sections showed GFP+ Myosin+ muscle fibers along with numerous GFP+ hematopoietic cells in damaged muscle. We also found myogenin+GFP+ cells like myoblasts in very low number. These phenomena were temporal and GFP signals had dramatically reduced 30 days after transplantation. FISH analysis confirmed the GFP-DNAs in the nuclei of muscle fibers. These results suggested that most of GFP+HSCs fused with myofibers and participated in regeneration of damaged muscles, and a very few HSCs can differentiate into myoblast like cells expressing myogenin. After 6 months, GFP+ fibers could be hardly detected but GFP+c-Met+ mononuclear cells were located beneath the laminin+ basal lamina. Single fiber cultures from these mice showed proliferation of GFP+ fibers. These results suggested that HSC-derived cells settled beneath the basal lamina like satellite cells and might acquired the satellite cell activity.

Estimating human hematopoietic stem cell kinetics using granulocyte telomere lengths

To study in vivo behavior of hematopoietic stem cells (HSC). Behavior of HSC is difficult to study because one cannot observe and track cells within the marrow microenvironment. Therefore, information must be obtained from indirect means, such as competitive repopulation assays or surrogate studies, such as observations of telomere shortening in granulocytes. In this article, we use granulocyte telomere length data and a novel approach, stochastic simulation, to derive replication rates of HSC. The approach is first applied to cats and then to humans. Human HSC replicate infrequently, on average once per 45 weeks (range: once per 23 to once per 67 weeks). This rate is substantially slower than the average replication rates estimated for murine (once per 2.5 weeks) and feline (once per 8.3-10 weeks) HSC in vivo.

Molecular signatures of proliferation and quiescence in hematopoietic stem cells.

Stem cells resident in adult tissues are principally quiescent, yet harbor enormous capacity for proliferation to achieve self renewal and to replenish their tissue constituents. Although a single hematopoietic stem cell (HSC) can generate sufficient primitive progeny to repopulate many recipients, little is known about the molecular mechanisms that maintain their potency or regulate their self renewal. Here we have examined the gene expression changes that occur over a time course when HSCs are induced to proliferate and return to quiescence in vivo. These data were compared to data representing differences between naturally proliferating fetal HSCs and their quiescent adult counterparts. Bioinformatic strategies were used to group time-ordered gene expression profiles generated from microarrays into signatures of quiescent and dividing stem cells. A novel method for calculating statistically significant enrichments in Gene Ontology groupings for our gene lists revealed elemental subgroups within the signatures that underlie HSC behavior, and allowed us to build a molecular model of the HSC activation cycle. Initially, quiescent HSCs evince a state of readiness. The proliferative signal induces a preparative state, which is followed by active proliferation divisible into early and late phases. Re-induction of quiescence involves changes in migratory molecule expression, prior to reestablishment of homeostasis. We also identified two genes that increase in both gene and protein expression during activation, and potentially represent new markers for proliferating stem cells. These data will be of use in attempts to recapitulate the HSC self renewal process for therapeutic expansion of stem cells, and our model may correlate with acquisition of self renewal characteristics by cancer stem cells.

Emergent Properties of Feedback Regulation and Stem Cell Behavior in a Granulopoiesis Model as a Complex System

To identify the internally generative theoretical relationship between microscopic mechanisms and the macroscopic behavior of hematopoietic processes as a complex system, a computer simulation of granulopoiesis was exploited by developing a cellular automaton (CA) model. Hematopoietic stem cells (HSCs) distribute themselves to proliferate and differentiate in a three-dimensional analytical space. The number of mitotic events of the cells in a proliferating phase, the transit times (T) of each of 15 differential stages progressing from a HSC to a mature cell, the duplication times ( T dup ) of HSCs, and the neighborhood rules for cell proliferation were all incorporated as analytical parameters in this space. Homeostatic granulopoiesis originating from a single HSC was successfully achieved. An important part is the stabilization of cell production induced by way of negative feedback following external perturbation of the peripheral granulocyte numbers. Single-cell kinetic analyses describe the behavior of differentiating cells and HSCs as fluctuating their T and self-renewal time ( T dup ) in response to the feedback dynamics. Stochastic cell divisions of HSCs were recruited in a transitional manner resulting in the generation of a regulatory effect on the differentiation--commitment processes. The concept that local cellular interaction produces global dynamics in a granulopoietic system was reified by CA modeling. This approach will provide a framework for analyzing the behavior of HSCs and enable an understanding of the abnormal kinetics of hematopoietic diseases.

Myeloid-lymphoid initiating cells (ML-IC) are highly enriched in the rhodamine-c-kit(+)CD33(-)CD38(-) fraction of umbilical cord CD34(+) cells.

The study of hematopoietic stem cells (HSC) is limited by lack of specific markers for HSC. Rhodamine 123 (Rho) is one of the substrates of P-glycoprotein (Pgp), and the presence of active Pgp can be shown by the efflux of Rho. Rho can also be used to measure the mitochondrial transmembrane potential (energy state) of a cell. We reasoned that selection of hematopoietic progenitors using a combination of Rho efflux and phenotypic markers might be superior to use of phenotypic markers alone. We used the myeloid-lymphoid initiating cell (ML-IC) assay as functional measure of primitive progenitors. Umbilical cord blood CD34(+)CD33(-)CD38(-), CD34(+)CD33(-)CD38(-)Rho(-), and CD34(+)CD33(-)CD38(-)Rho(-)c-kit(+) cells were sorted singly onto AFT024 feeders to assess their capacity to become ML-IC. The frequency of ML-IC in CD34(+)CD33(-)CD38(-)Rho(-) cells was significantly higher (15 +/- 0.4%) than that in CD34(+)CD33(-)CD38(-) cells (6.2 +/- 0.9%, p < 0.05). However, the frequency of long-term culture-initiating cells (LTC-IC) (17 +/- 3% vs 12 +/- 1.5%) and natural killer culture-initiating cells (NK-IC) (25 +/- 3% vs 20 +/- 4%) was similar in the two populations. Following the treatment of CD34(+)CD33(-)CD38(-)Rho(-) cells with verapamil, which blocks Pgp function, no increase in ML-IC was detected compared with CD34(+)CD33(-)CD38(-) cells (6 +/- 0.7%), suggesting that differences in the energy state, which is reflected by Rho staining after verapamil treatment, cannot be used as a criterion to identify human HSC. Further selection of CD34(+)CD33(-)CD38(-)Rho(-) cells based on expression of c-kit significantly increased the frequency of ML-IC, LTC-IC and NK-IC by 1.75-, 1.3-, and 1.8-fold, respectively. Combining the function of Pgp and phenotypic features of hematopoietic progenitors enriches the frequency of cord blood ML-IC to greater than 25%. Use of such enriched populations will allow us to characterize the biological behavior of human HSC.

Telomeres, X‐Inactivation Ratios, and Hematopoietic Stem Cell Transplantation in Humans: A Review

The marrow repopulating hematopoietic stem cells (HSCs) in an auto- or allograft represent a small fraction of the normal complement of HSCs, yet are required to reconstitute hematopoiesis and sustain it for the lifetime of the recipient. Such a burden imposes a "replicative stress" upon hematopoietic stem/progenitor cells. The finding of accelerated telomere shortening in hematopoietic stem cell transplant (HSCT) recipients raised the specter of accelerated hematopoietic aging. Here, we review the HSCT telomere literature and other studies of surrogate markers of HSC behavior conducted in human HSCT recipients. We present a paradigm for posttransplant hematopoietic reconstitution and speculate on the fate of HSCs in the human transplant setting.