Hematopoietic Progenitor and Stem Cell Regulation during Development: Hypoxia and Niches

In a healthy adult person, almost 1011 - 1012 new blood cells are generated daily in order to maintain the steady state in peripheral circulation (Paul, 2008). To this end, a high level of self-renewal and differentiation of Hematopoietic stem cells (HSCs) is required. HSCs have the unique ability of self-renewal to maintain the stem cell pool. They also differentiate to more committed progenitors which produce all mature blood cell lineages (Lemischka, 1992; Medvinsky and Dzierzak, 1998; Orkin, 2000). In general, there are two main branches in the adult hematopoietic hierarchy: The lymphoid branch is derived from common lymphoid progenitors that mature to B-cells and T-cells. Lymphocytes are involved in the adaptive immune system. The myeloid branch is derived from common myeloid progenitor cells that give rise to more lineage-restricted precursors. These progenitors differentiate into the following cell types: erythrocytes, the most abundant terminally differentiated cells in the blood, which are required for oxygen transport, as well as megakaryocytes, involved in blood clotting and granulocytes and macrophages, which are involved in innate immunity. Recent insights into understanding HSC regulation have facilitated HSC clinical therapies and trials for hematological disorders. Despite progress in this field, there remain difficulties in ex vivo expansion of HSCs on a scale that ensures efficient regeneration of blood system. Overcoming this challenge calls for a more information on the intrinsic and extrinsic factors that regulate development, maintenance, and function of HSCs

HIF1α is a regulator of hematopoietic progenitor and stem cell development in hypoxic sites of the mouse embryo

Hypoxia affects many physiologic processes during early stages of mammalian ontogeny, particularly placental and vascular development. In the adult, the hypoxic bone marrow microenvironment plays a role in regulating hematopoietic stem cell (HSC) function. HSCs are generated from the major vasculature of the embryo, but whether the hypoxic response affects the generation of these HSCs is as yet unknown. Here we examined whether Hypoxia Inducible Factor1-alpha (HIF1α), a key modulator of the response to hypoxia, is essential for HSC development. We found hypoxic cells in embryonic tissues that generate and expand hematopoietic cells (aorta, placenta and fetal liver), and specifically aortic endothelial and hematopoietic cluster cells. A Cre/loxP conditional knockout (cKO) approach was taken to delete HIF1α in Vascular Endothelial-Cadherin expressing endothelial cells, the precursors to definitive hematopoietic cells. Functional assays show that HSC and hematopoietic progenitor cells (HPCs) are significantly reduced in cKO aorta and placenta. Moreover, decreases in phenotypic aortic hematopoietic cluster cells in cKO embryos indicate that HIF1α is necessary for generation and/or expansion of HPCs and HSCs. cKO adult BM HSCs are also affected under transplantation conditions. Thus, HIF1α is a regulator of HSC generation and function beginning at the earliest embryonic stages

Hypoxia and HIFs in regulating the development of the hematopoietic system

Many physiologic processes during the early stages of mammalian ontogeny, particularly placental and vascular development, take place in the low oxygen environment of the uterus. Organogenesis is affected by hypoxia inducible factor (HIF) transcription factors that are sensors of hypoxia. In response to hypoxia, HIFs activate downstream target genes - growth and metabolism factors. During hematopoietic system ontogeny, blood cells and hematopoietic progenitor/stem cells are respectively generated from mesodermal precursors, hemangioblasts, and from a specialized subset of endothelial cells that are hemogenic. Since HIFs are known to play a central role in vascular development, and hematopoietic system development occurs in parallel to that of the vascular system, several studies have examined the role of HIFs in hematopoietic development. The response to hypoxia has been examined in early and mid-gestation mouse embryos through genetic deletion of HIF subunits. We review here the data showing that hematopoietic tissues of the embryo are hypoxic and express HIFs and HIF downstream targets, and that HIFs regulate the development and function of hematopoietic progenitor/stem cells

Role of metastasis-associated lung adenocarcinoma transcript-1 (MALAT-1) in pancreatic cancer - Fig 6

<p><b>(A) Fold-change in MALAT-1 gene expression in knockout mice as compared to the wild type.</b> (B) Sp1, Sp3, Sp4 and c-Myc expression in homozygous floxed p53/Kras^GD12 mice. (C) Survival of homozygous floxed p53L/L: Kras^GD12:p48Cre+/- mice expression MALAT-1 (+) or with loss of MALAT-1 (-/+). (D) Survival of heterozygous floxed p53L/+: Kras^GD12:p48Cre+/- mice expression MALAT-1 (+) or with loss of MALAT-1 (-/+). (E) Histology analysis of tumor samples from different strains of mice; images were 200X and 600X (for corner inserts). p-Values for significant differences in (C) and (D) were 0.39 and 0.26, respectively.</p

Role of metastasis-associated lung adenocarcinoma transcript-1 (MALAT-1) in pancreatic cancer - Fig 5

<p><b>(A) The structure of CDDO-Me and CF</b>_<b>3</b><b>DODA-Me.</b> Panc1 cells were treated with different concentrations of CDDO-Me or in combination of GSH, CF₃DODA-Me or in combination with GSH, and the changes of different proteins (B) and MALAT-1 expression (C) were determined by western blot and real time PCR, respectively. (D) Panc1 cells were transfected with siSp1/3/4 or siCtrl, and the MALAT-1 RNA expression was determined by real time PCR. Significant (p<0.05) changes are indicated (*) or (**).</p

Gene regulation by MALAT-1.

<p>(A) Panc1 cells were transfected with oligonucleotides (siMALAT-1#1/siMALAT-1#2) and expression of MALAT-1 was determined by real time PCR. (B) Panc1 cells were transfected with siMALAT-1 or siCtrl and gene expression was analyzed using Human HT-12 v4 expression beadchip (Illumina, Inc.) array. (C) The effects of siMALAT-1 on different function categories and the predicted activation state of cell proliferation, death and movement after HOTTIP knockdown were determined by IPA. (D) Pathway analysis. Causal IPA was used to analyze the p-values and Z-scores for cell movement proliferation and cell death after MALAT-1 knockdown by RNA interference.</p

Effects of MALAT-1 in pancreatic cell proliferation, cell cycle, apoptosis, migration and invasion.

<p>(A) MALAT-1 knockdown by RNAi in Panc1, MiaPaCa2 inhibited cell growth. (B) The effect of siMALAT-1 (knockdown) on cell cycle progression in Panc1 and MiaPaCa2 cells was determined by FACS analysis. (C) The apoptotic cells were quantified using FACS analysis and induction of PARP cleavage was determined by western blot analysis. MALAT-1 knockdown reduced cell migration (D) and cell invasion (E) as determined by scratch assay and Boyden chamber assay, respectively. Significant (p<0.05) changes are indicated (*).</p