O22. Distinct roles of different nitric oxide synthase isoforms in regulating hematopoietic stem cell function in vivo

Nitric Oxide ◽  
2006 ◽  
Vol 14 (4) ◽  
pp. 7 ◽  
Author(s):  
Youzhong Yuan ◽  
Edith Tzeng ◽  
Timothy R. Billiar
Keyword(s):  
Nitric Oxide ◽  
Stem Cell ◽  
Nitric Oxide Synthase ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Hematopoietic Stem ◽  
Oxide Synthase

Loss of FancC Function Results in Decreased Hematopoietic Stem Cell Repopulating Ability

Blood ◽  
1999 ◽  
Vol 94 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Laura S. Haneline ◽  
Troy A. Gobbett ◽  
Rema Ramani ◽  
Madeleine Carreau ◽  
Manuel Buchwald ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Genetic Disorder ◽  
Test Cell ◽  
Hematopoietic Stem ◽  
Murine Homologue ◽  
Complex Genetic Disorder

Fanconi anemia (FA) is a complex genetic disorder characterized by progressive bone marrow (BM) aplasia, chromosomal instability, and acquisition of malignancies, particularly myeloid leukemia. We used a murine model containing a disruption of the murine homologue ofFANCC (FancC) to evaluate short- and long-term multilineage repopulating ability of FancC −/− cells in vivo. Competitive repopulation assays were conducted where “test”FancC −/− or FancC +/+ BM cells (expressing CD45.2) were cotransplanted with congenic competitor cells (expressing CD45.1) into irradiated mice. In two independent experiments, we determined that FancC −/− BM cells have a profound decrease in short-term, as well as long-term, multilineage repopulating ability. To determine quantitatively the relative production of progeny cells by each test cell population, we calculated test cell contribution to chimerism as compared with 1 × 105 competitor cells. We determined that FancC −/− cells have a 7-fold to 12-fold decrease in repopulating ability compared with FancC +/+cells. These data indicate that loss of FancC function results in reduced in vivo repopulating ability of pluripotential hematopoietic stem cells, which may play a role in the development of the BM failure in FA patients. This model system provides a powerful tool for evaluation of experimental therapeutics on hematopoietic stem cell function.

scholarly journals New genetic tools for the in vivo study of hematopoietic stem cell function

2018 ◽  
Vol 61 ◽  
pp. 26-35 ◽  
Author(s):  
Samik Upadhaya ◽  
Boris Reizis ◽  
Catherine M. Sawai
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
In Vivo Study ◽  
Hematopoietic Stem ◽  
Genetic Tools

Characterization of Hematopoietic Stem Cell Function and Sensitivity in the Homologous Recombination Deficient Exonuclease1mut Mouse Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1293-1293
Author(s):  
Amar Desai ◽  
Yulan Qing ◽  
Stanton L. Gerson
Keyword(s):  
Dna Repair ◽  
Stem Cell ◽  
Homologous Recombination ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Strand Break ◽  
Hematopoietic Stem ◽  
Quiescent Hscs

Abstract Abstract 1293 Hematopoietic stem cell (HSC) maintenance is essential for sustained longevity and tissue function. The HSC population has lifelong self-renewing capabilities and gives rise to subsets of multipotent progenitor cells, and in turn a progeny of terminally differentiated mature cells consisting of all subtypes of the myeloid and lymphoid lineages. Long term reconstituting HSCs are necessary to replace these differentiated cells after losses caused by normal degradation or damage accumulation, with failure to replenish these stores being linked to a variety of human pathogeneses as well as aging phenotypes. HSC populations require functional DNA repair pathways in order to maintain their reconstitution capabilities but little is known about the pathways involved or the mechanism of regulation. While the majority of HSCs are quiescent at steady state, endogenous or exogenous stress can force these cells into proliferation, and previous evidence has suggested that the HSC reliance on DNA repair changes with this mobilization. Quiescent HSCs are believed to depend on non-homologous end joining (NHEJ) for repair but prior literature has shown that once forced into cycle, the DNA repair dependency shifts and is shared between homologous recombination (HR) and NHEJ. We use Exo1 deficiency as a model for homologous recombination loss in mice and demonstrate in vivo that HR is dispensable in quiescent HSCs. This is in contrast to loss of the complementary double strand break repair pathway NHEJ which has been shown to result in severe defects in HSC function. However when we force mobilize HSCs into cycle in vivo using the anti metabolite 5-fluorouracil we are able to demonstrate that the HR defects become detrimental to the animal as shown by increased cellular IR sensitivity and subsequent animal death. Additionally we use competitive repopulation studies to show that indeed the Exo1mut HSC population is more radiation sensitive after forced mobilization. This work begins to elucidate the consequences of the loss of homologous recombination in hematopoietic stem cells as well as the interplay between cell cycle status and DNA repair dependency. Disclosures: No relevant conflicts of interest to declare.

Overexpression of Rheb2 Has Diverse Effects on Hematopoietic Progenitor and Stem Cell Functions.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1274-1274
Author(s):  
Timothy B. Campbell ◽  
Giao Hangoc ◽  
Wen Tao ◽  
Hal E. Broxmeyer
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Cell Lines ◽  
Progenitor Cell ◽  
Cell Function ◽  
Hematopoietic Stem ◽  
Cell Functions ◽  
Empty Vector ◽  
Delayed Growth

Abstract Determining which intracellular signaling pathways are important for hematopoietic stem and progenitor cell functions is important for therapeutic uses of these cells. The intracellular serine/threonine kinase mammalian target of rapamycin (mTOR) is important for regulation of the growth, proliferation, survival and differentiation of various cell types in the body, including normal hematopoietic and blood cancer cells. We chose to study the protein Ras homologue enriched in brain-2 (Rheb2), a known activator of mTOR signaling, because there is evidence it is preferentially expressed in highly purified mouse (m) hematopoietic stem cell populations (Ivanova et al. Science, 298(5593):601–04, 2002). Rheb2 is one of two members of the Rheb small GTPase family of proteins known to activate mTOR by mechanisms which are not fully elucidated, but that are regulated by Akt and the tuberous sclerosis complex (TSC1/2). We overexpressed mouse Rheb2 protein in both primary mouse bone marrow (mBM) cells and two mouse hematopoietic cell lines (BaF3 and 32D) and determined effects on progenitor cell colony-formation, delayed IL-3 driven proliferation in cell lines and hematopoietic stem cell function in an in vivo competitive stem cell repopulation assay. Overexpression of Rheb2 in retrovirally transduced mBM cells significantly increased the colony-forming ability of the cells in response to growth factors. The positive effect of Rheb2 was most pronounced on granulocyte-macrophage (CFU-GM) progenitors (greater than two-fold increase over empty vector transduced cells) and was persistent in cells expanded in liquid culture up to one week after FACS sorting of transduced cells. Additionally, Rheb2 overexpression significantly increased the ability of mBM cells to form CFU-GM colonies under conditions of delayed-growth factor addition (at 24 and 48 hours after plating), suggesting a positive role for Rheb2 in the survival of these cells. Interestingly, addition of the pro-survival chemokine SDF-1/CXCL12 to the delayed-growth factor colony assay plates enhanced the survival of the empty vector transduced cells but did not further enhance the survival of the Rheb2 overexpressing cells. Using two IL-3 dependent hematopoietic cell lines, BaF3 and 32D, to further assess Rheb2 function and signaling, we found overexpression of Rheb2 enhanced proliferation after delayed addition of IL-3. These changes correlated with an increase in mTOR signaling in Rheb2 transduced cells. In order to evaluate the effect of Rheb2 overexpression on hematopoietic stem cell function, an in vivo competitive repopulation assay was performed. Transduced donor and non-transduced competitor cells were pooled at two ratios (1:1 and 0.4:1 donor to competitor) and injected into lethally irradiated recipients. Transduced cells were followed in the recipients by measuring GFP+ cell chimerism. In contrast to the data on progenitor cell function, transduced Rheb2 cells had significantly decreased competitive repopulation in the recipient mice (greater that 3-fold decrease compared to empty vector). This suggests that Rheb2 overexpression may negatively affect HSC homing, stable engraftment and/or self-renewal. Overall, these studies demonstrate that overexpression of Rheb2 positively enhances progenitor cell functions such as colony-formation, while negatively affecting stem cell competitive repopulation, thereby providing a possible regulatory mechanistic link between the flow from the stem to progenitor cell compartment.

Signaling via Smad2 and Smad3 is dispensable for adult murine hematopoietic stem cell function in vivo

2017 ◽  
Vol 55 ◽  
pp. 34-44.e2 ◽  
Author(s):  
Matilda Billing ◽  
Emma Rörby ◽  
Maria Dahl ◽  
Ulrika Blank ◽  
Silja Andradottír ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Hematopoietic Stem ◽  
Murine Hematopoietic Stem Cell

scholarly journals The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function

2015 ◽  
Vol 113 (1) ◽  
pp. E51-E60 ◽  
Author(s):  
Yue Gu ◽  
Amanda E. Jones ◽  
Wei Yang ◽  
Shanrun Liu ◽  
Qian Dai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Ubiquitin Ligase ◽  
Cell Function ◽  
Lymphoid Organ ◽  
Histone H2a ◽  
Hematopoietic Stem

Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation.

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