376 research outputs found
Identification of Resident and Circulating Endothelial Stem Cells
No full textBlood vessels and circulating blood contain rare immature endothelial cells that display in vitro clonal proliferative potential and in vivo vessel forming ability. However their precise location, origin, surface marker and molecular determinants have yet to be precisely defined. In this research body of work, we have identified ABCG2, an ATP binding cassette drug transporter that is expressed by stem cells of many lineages, to label vascular endothelial stem cells (VESC) and circulating endothelial stem cells (CESC). During development, ABCG2 expressing VESC are distributed in arterial, venous, and capillary vessels of multiple tissues including heart, lung, bone marrow and retina. They possess clonal colony forming potential in vitro and contribute to the growth of arteries, veins and capillaries in vivo. Steady state adult tissues also contain VESC that retain colony forming potential, though their frequency is decreased. In human umbilical vessels, ABCG2+ VSEC represent about 1% of umbilical cord vessel EC and showed higher colony forming potential than ABCG2- EC. In addition, CESC that could form EC colonies in vitro were identified from neonatal murine peripheral blood. About 30% of CESC were labeled by ABCG2. Lineage tracing experiments using hematopoietic (Flk2Cre) and EC (Tie2ERTCre) specific mice showed CESC were derived from vascular EC, not hematopoietic cells. CESC could participate (at a single cell level) in vessel formation in vivo in gel transplantation model. Furthermore, after transplantation, CESC retained secondary colony forming potential in formed blood vessels. Finally, we show that Abcg2 not only labels, but is also critical for the emergence/maintenance of VESC and the production of CESC. These findings provide a solid foundation to identify the critical roles of endothelial stem cells in vascular development, homeostasis and repair
IMPAIRED FUNCTION OF FANCONI ANEMIA TYPE C DEFICIENT MACROPHAGES
No full textIndiana University-Purdue University Indianapolis (IUPUI)Fanconi anemia (FA) is a genetic disorder characterized by bone marrow (BM) failure. Previous studies suggest that FA patients exhibit alterations in immunologic function. However, it is unclear whether the immune defects are immune cell autonomous or secondary to leucopenia from evolving BM failure. The aim of the current study was to determine whether FA type C deficient (Fancc-/-) macrophages exhibit impaired function and contribute to an altered inflammatory response. In this study, primary peritoneal macrophage function and the inflammatory response of Fancc-/- immune cells after in vivo intraperitoneal (IP) administration of lipopolysaccharide (LPS) were assessed. Fancc-/- peritoneum exhibit normal macrophage distribution at baseline. However, Fancc-/- macrophages exhibit reduced adhesion both on fibronectin and endothelial cells, impaired migration toward monocyte chemotactic protein-1 (MCP-1) and macrophages-colony stimulating factor (M-CSF), and altered phagocytosis of E.coli and ImmunoglobulinG (IgG)-labeled latex beads compared to WT. An altered F-actin reorganization and impaired activation of RhoA were observed in Fancc-/- macrophages. After single LPS injection IP, Fancc-/- mice exhibited decreased macrophage recruitment, reduced peripheral inflammatory monocytes and impaired myeloid colony formation in presence of M-CSF. Upon M-CSF stimulation, Fancc-/- BM derived macrophages (BMDM) showed a decreased phosphorylation of AKT and ERK compared to WT, leading to reduced proliferation. Collectively, these data suggest that Fancc-/- macrophages and subsequent defects in adhesion, migration, phagocytosis, and recruitment in vivo. These data also support a Fancc-/- macrophage cells autonomous defect predisposing to an altered inflammatory response
The Origin of Juvenile Myelomonocytic Leukemia: Insights from Developmental Hematopoiesis
No full textHematopoiesis proceeds through three developmental phases, each with a unique and indispensable function. The individual roles of these phases in the pathogenesis of blood disorders is unknown. We have adapted murine lineage trace models to identify the relative contributions of embryonic, fetal, and adult hematopoietic phases to the origin of Juvenile Myelomonocytic Leukemia. We hypothesized that the fetal phase would have the most pronounced contribution to the development of JMML, a pediatric myeloproliferative disorder whose disease-initiating somatic mutations occur in utero. Progenitors expressing PTPN11E76K from all three waves were growth hypersensitive to GM-CSF due to hyperactive RAS-ERK signaling. However, fulminant myeloproliferation was only seen in fetal and adult cohorts. We observed equal disease severity in FLT3Cre; PTPN11 E76K; ROSA26mTmG and CSF1R-MCM; PTPN11E76K; ROSA26YFP cohorts, which had high and low mutant allele frequencies, respectively. This led to the revelation that all progenitors in the BM niche of mutant animals have equal growth hypersensitivity and RAS-ERK hyperactivation due to non-cell autonomous effects of PTPN11E76K. We further identified that FLT3Cre has hematopoietic-restricted expression, and thereby circumvented morbidity from PTPN11E76K expression in endothelial and stromal cells. This led us to hypothesize that FLT3Cre; KrasG12D; ROSA26mTmG would be the first faithful model of JMML to express this disease-initiating mutation. Indeed, FLT3Cre; KrasG12D mice were born at expected Mendelian ratio and showed normal weight gain to 2 weeks of age. Thereafter, they acquired defining features of JMML including monocytosis, anaemia, thrombocytopenia, and hepatosplenomegaly. All FLT3Cre; KrasG12D mice succumb to a JMML-like disease, which was propagated following transplantation. This is in contrast with CSF1R-MCM; KrasG12D; ROSA26YFP mice, in which low mutant allele frequencies in either fetal or adult HSCs uniformly resulted in T-ALL. Our models reveal previously underappreciated features of JMML including an expansion of dendritic cells and a pronounced defect in T-lymphocyte development. We are the first to demonstrate non-cell autonomous effects of hematopoietic-restricted PTPN11E76K expression. Most importantly, we have shown that both the spatial and the temporal origin of JMML-initiating mutations will affect disease manifestations. Each of our findings suggest novel strategies to treat this intractable disease
A Bipotent Mesoderm Subset Identified via Colony-Forming Assay
No full textThe use of an in vitro colony-forming assay has previously led to identification of a mesoderm-derived bipotent hemangioblast from differentiating hESCs. In this issue of Cell Stem Cell, Vodyanik et al. have used a similar approach to herald the presence of a bipotent mesoderm-derived precursor for mesenchymal stem and endothelial cells, termed the mesenchymoangioblast
Endothelial stem and progenitor cells (stem cells): (2017 Grover Conference Series)
No full textThe capacity of existing blood vessels to give rise to new blood vessels via endothelial cell sprouting is called angiogenesis and is a well-studied biologic process. In contrast, little is known about the mechanisms for endothelial cell replacement or regeneration within established blood vessels. Since clear definitions exist for identifying cells with stem and progenitor cell properties in many tissues and organs of the body, several groups have begun to accumulate evidence that endothelial stem and progenitor cells exist within the endothelial intima of existing blood vessels. This paper will review stem and progenitor cell definitions and highlight several recent papers purporting to have identified resident vascular endothelial stem and progenitor cells
Endothelial progenitor cell: a blood cell by many other names may serve similar functions
No full textThe first reports of circulating cells that displayed the capacity to repair and regenerate damaged vascular endothelial cells as progenitor cells for the endothelial lineage (EPC) were met with great enthusiasm. However, the cell surface antigens and colony assays used to identify the putative EPC were soon found to overlap with those of the hematopoietic lineage. Over the past decade, it has become clear that specific hematopoietic subsets play important roles in vascular repair and regeneration. This review will provide some overview of the hematopoietic hierarchy and methods to segregate distinct subsets that may provide clarity in identifying the proangiogenic hematopoietic cells. This review will not discuss those circulating viable endothelial cells that play a role as EPC and are called endothelia colony-forming cells. The review will conclude with identification of some roadblocks to progress in the field of identification of circulating cells that participate in vascular repair and regeneration
Effects of Altering Cell Proliferation on Hematopoietic Stem and Progenitor Cell Function
No full textIndiana University-Purdue University Indianapolis (IUPUI)Cell cycle checkpoints guarantee movement through the cell cycle in an appropriate manner. The spindle assembly checkpoint (SAC) ensures the proper segregation of chromosomes into daughter cells during mitosis. Mitotic arrest deficiency 2 (Mad2), a member of the mitotic checkpoint proteins, appears to be crucial for generating the wait anaphase signal to prevent onset of anaphase. We first studied the SAC in hematopoietic stem cells (HSC) to ensure that it was functional. Our previous studies found that prolonged SAC activation was uncoupled from apoptosis initiation in mouse and human embryonic stem cells (ESC). We found that upon treatment with a microtubule-destabilizing agent, HSC arrested in M-phase and subsequently initiated apoptosis. Thus unlike ESC, HSC exhibit coupling of prolonged SAC activation with apoptosis. We studied the effects of Mad2+/- on in vivo recovery of bone marrow HPC from cytotoxic effects and also effects of cytostatic agents on HPC growth in vitro using Mad2-haploinsufficient (Mad2+/-) mice. We found that Mad2+/- HPCs were protected from the cytotoxic effects of cytarabine (Ara-C), a cycle specific agent, consistent with Mad2+/- HPCs being in a slow or non-cycling state. Mad2 haploinsufficiency did not affect recovery of functional HPC after treatment with cyclophosphamide or high sub-lethal dose irradiation, both non-cycle specific agents. There were no differences in immunophenotype defined HSCs in Mad2+/- and Mad2+/+ mice, data confirmed by functional HSC competitive repopulation assays. To better understand the role of Mad2 in HPC, E3330, a cytostatic agent, was used to assess the redox function of Ape1/Ref-1, and colony formation in vitro was examined under normoxic and lowered O2 tension. Mad2+/- HPCs were less responsive to E3330 than Mad2+/+ HPCs, and E3330 was more effective under lowered O2 tension. Mad2+/- HPCs did not exhibit enhanced growth in lowered oxygen tension, in contrast to Mad2+/+ HPCs. Our studies have unexpectedly found that Mad2 haploinsufficiency is protective from the cytotoxic effects of a cycle specific DNA synthesis agent in vivo, and Ape1/Ref-1 inhibitor in vitro
Mutant P53 in pre-leukemic hematopoietic stem cells and the pathogenesis of Myelodysplastic Syndrome
No full textIndiana University-Purdue University Indianapolis (IUPUI)Myelodysplastic syndrome (MDS) is a clonal disease arising from mutated
hematopoietic stem cells (HSCs). MDS stem cells originate from pre-leukemic
HSCs, which have enhanced competitive advantage over wild-type (WT) HSCs
but normal differentiation capacity. Recently, acquired somatic gain-of-function
(GOF) TP53 mutations were identified in the blood of aged healthy individuals as
well as in patients with MDS. However, the role of GOF TP53 mutations in clonal
hematopoiesis and the pathogenesis of MDS is largely unknown.
Based upon our previous studies and clinical findings, I hypothesized that
GOF mutant p53 drives the development of pre-leukemic HSCs with enhanced
competitive advantage, leading to clonal expansion and the pathogenesis of
MDS. To test my hypothesis, I examined HSC behaviors in young p53+/+ and
p53R248W/+ mice. I discovered that p53R248W enhances the repopulating potential
of HSCs without affecting terminal differentiation. I also found that GOF mutant
p53 protects HSCs from genotoxic stress and promotes their expansion. To
investigate the role of mutant p53 in the pathogenesis of hematological
malignancies, I monitored disease development in p53+/+ and p53R248W/+ mice
and observed that some mutant p53 mice develop MDS during aging. Therefore,
I demonstrated that GOF mutant p53 enhances the repopulating potential of HSCs and drives the development of pre-leukemic HSCs, predisposing aged mutant p53 mice to MDS development.
Mechanistically, I found that mutant p53 increases the chromatin accessibility to genes important for HSC maintenance, including pluripotent gene Sox2 and chemokine gene Cxcl9. By performing biochemical experiments, I discovered that GOF mutant p53, but not WT p53, interacts with histone methyltransferase EZH2 and enhances histone H3 lysine 27 trimethylation (H3K27me3) at genes, including Mef/Elf4 and Gadd45g, that negatively regulate HSC self-renewal.
Collectively, these findings demonstrated that GOF mutant p53 drives pre-leukemic HSC development through modulating epigenetic pathways. Thus, our studies have uncovered novel mechanistic and functional links between GOF mutant p53 and epigenetic regulators in pre-leukemic HSCs. This research may identify epigenetic regulator EZH2 as a novel target for the prevention and treatment of MDS patients with TP53 mutations
SIRT1 DEFICIENCY COMPROMISES MOUSE EMBRYONIC STEM CELL DIFFERENTIATION, AND EMBRYONIC AND ADULT HEMATOPOIESIS IN THE MOUSE
No full textIndiana University-Purdue University Indianapolis (IUPUI)SIRT1 (Sirtuin 1) is a founding member of a family of seven proteins and histone deacetylases. It is involved in cellular resistance to stress, metabolism, differentiation, aging, and tumor suppression. SIRT1-/- mice demonstrate embryonic and postnatal development defects. We examined hematopoietic and endothelial cell differentiation of SIRT1-/- mouse embryonic stem (mES) cells in vitro, and hematopoietic progenitors in SIRT1+/+, SIRT1+/-, and SIRT1-/- mice. SIRT1-/- ES cells exhibited markedly delayed/immature formation of blast colony-forming cells (BL-CFCs). When individual blast colonies were analyzed for hematopoietic and endothelial potential, replated SIRT1-/- BL-CFC possessed limited hematopoietic potential, whereas endothelial potential was essentially unaltered. The ability of SIRT1-/- ES cells to form primitive erythroid progenitors was not only delayed but greatly decreased. Moreover, after differentiation of SIRT1-/- mES cells, there were also significant decreases in granulocyte-macrophage (CFU-GM) and multipotential (CFU-GEMM) progenitor cells. Differentiation delay/defects were associated with delayed capacity to switch off Oct4, Nanog and Fgf5, decreased β-H1 globin, β-major globin, and Scl gene expression and reduced activation of the Erk1/2 pathway upon SIRT1-/- ES cell commitment. Reintroduction of WT SIRT1 into SIRT1-/- cells partially rescued the primitive erythroid progenitor formation of SIRT1-/- cells and the expression of hemoglobin genes, Hbb-bh1 and Hbb-b1, suggesting that the defect of hematopoietic commitment is due to deletion of SIRT1, and not to genetic drifting of SIRT1-/- cells. To confirm the requirement for SIRT1 for normal development of hematopoietic progenitor cells, we assessed embryonic and adult hematopoiesis in SIRT1+/+, SIRT1+/- and SIRT1-/- mice. Yolk sacs from SIRT1 mutant embryos generated fewer primitive erythroid precursors compared to wild-type (WT) and heterozygous mice. Moreover, knockout of SIRT1 decreased primary bone marrow hematopoietic progenitor cells (HPCs) in 5 week and 12 month old mice, which was especially notable at lower (5%) O2 tension. In addition these progenitors survived less well in vitro under conditions of delayed growth factor addition. Taken together, these results demonstrate that SIRT1 plays a role in ES cell hematopoietic differentiation and mouse hematopoiesis
- …
