Fetal Liver Hematopoietic Stem Cells Research Articles

An anti-inflammatory effect of murine fetal liver cells in BALB/c mouse contact hypersensitivity model

Anti-inflammatory effects of murine fetal liver (FL) cells were studied using BALB/c mouse contact hypersensitivity (paw edema) model. Paw weight differences, lymphatic organ weights, hematological and histological indices as well as proinflammatory (TNF-α) and anti-inflammatory (IL-10) cytokine levels in sera were evaluated. Immunophenotyping revealed that both murine FL homogenate cells (HC) and FL hematopoietic stem cells (HSC) express CD117 and CD38 surface markers. Single doses of 1×106 cells/mouse and 2×106 cells/mouse of FL HC as well as of FL HSC, when used separately, all statistically significantly (p<0.05) inhibited paw edema, but the lower dose was more effective and giving results similar to that of prednisolone. Either dose of FL HC or FL HSC studied had no significant influence on lymphatic organ weights; no significant changes were also observed in blood indices. The data of cytokine studies showed that TNF-α concentration in sera of mice treated with either FL HC or FL HSC at a dose of 1×106 cells/mouse was statistically significantly (p<0.001) lower than that of the control mice. A concentration of IL-10 was statistically significantly higher (p<0.01) in mice treated with a dose of 1×106 cells/mouse of FL HC but not with the same dose of FL HSC as compared to the control group. Histological examination revealed better effects of a dose of 1×106 cells/mouse of FL HC when compared with the same dose of FL HSC as in regard to reduction of edema thickness and cell infiltration.

Distinct Roles of Integrins α6 and α4 in Fetal Liver Hematopoietic Stem and Progenitor Cell Homing.

During development and after transplantation, intravenously injected hematopoietic stem and progenitor cells (HSPCs) selectively transmigrate through the sinusoidal walls into the bone marrow (BM) niches to engraft and reconstitute hematopoiesis. The rate of reconstitution following transplantation varies depending on the source of hematopoietic stem cells (HSCs) (To, et al 1992). However, the molecular pathways that control the homing of HSCs, in particular, of fetal HSCs are still not well understood. In the present study we studied the contribution of α6 and α4 integrins in homing of fetal liver HSPCs into adult BM by using function-blocking antibodies and an integrin α6 knockout mouse model. We found an ubiquitous expression of both integrin α6 and α4 receptors on fetal liver Lin−Sca-1+c-kit+ (LSK) HSPCs. Genetic ablation of integrin α6 resulted in reduced homing of fetal liver progenitors (HPCs) to BM of lethally irradiated adult recipients. In agreement with this, the integrin α6 antibody inhibited homing of fetal liver HPCs into BM and spleen. The role of integrin a6 in homing and engraftment of fetal liver HSCs was studied by a competitive repopulation assay by using integrin α6−/− or α6+/+ fetal liver cells. Absence of α6 integrin in fetal liver cells did not cause any engraftment defect or mobilization hypersensitivity as compared to wild-type cells. In agreement with this, anti-integrin α6 antibody did not either inhibit BM homing of short-term or long-term HSCs. In contrast, homing of fetal liver HSCs and HPCs to BM was virtually abrogated after treatment with integrin α4 antibody. Our results show that the α6 integrin receptors are functional during homing of fetal liver HPCs, but not multilineage repopulating HSC in vivo. Furthermore, we show the critical role of integrin α4 receptor for homing of both fetal liver HPCs and multilineage repopulating HSCs to BM, indicating distinct developmentally regulated functions for integrin α6 and α4 receptors during fetal hematopoiesis

The Wilms' Tumor Gene WT1 Is Over-Expressed in Immature Leukemia Cells but Not Necessary for Leukemia Development in Mouse Leukemia Models.

The Wilms tumor gene WT1, which is over-expressed in almost all leukemia, is one of the most promising targets for immunotherapy. To clarify which cells express WT1, we generated a knock-in reporter GFP mice (WT1GFP/+) and assayed for WT1 expression in normal and leukemic hematopoietic cells. In normal hematopoietic cells, WT1 was expressed in none of the long-term hematopoietic stem cells (HSCs) and very few (<1%) of multipotent progenitor cells. WT1 was expressed in 17.4% of megakaryocyte-erythrocyte progenitor cells and 4.1% of common myeloid progenitor cells, while the frequencies of WT1 expressing cells in mature granulocyte or lymphocyte were very low (~1%). In contrast, in murine leukemias induced by TEL/PDGFβR + AML1/ETO or BCR/ABL, WT1 was expressed in 40.5% or 38.9% of c-kit+lin-Sca-1+ (KLS) leukemia cells, which contained a subset, but not all, of transplantable leukemic stem cells (LSC). Furthermore, WT1 was not expressed fetal liver HSC population, mobilized HSCs by cyclophosphamide and granulocyte-colony stimulating factor, or HSCs stimulated with SCF, Flt3L, and IL3 in-vitro. These results suggest that WT1 expression in is not a surrogate marker of cell proliferation. To examine the function of WT1 in leukemogenesis, we used recombinant lentivirus to overexpress WT1. HSCs transduced with either empty or WT1-expressing lentivirus were transplanted into lethally irradiated recipients. The FACS analysis of BM cells at 4 months post transplant showed very low chimerism of WT1-expressing cells compared to control GFP-expressing cells. Moreover, differentiation block of WT1-expressing cells was not observed. These results indicate that WT1 does not induce proliferation or block differentiation in-vivo. Next, we tested whether leukemias could be induced from the WT1-deficient fetal liver hematopoietic cells. All of three mice transplanted with E13.5 WT1-defeicient fetal liver cells transduced with TEL/PDGFβR+AML1/ETO developed leukemia. Similarly, 3 out of 4 mice transplanted with p210BCR/ABL-transduced WT1-deficient fetal liver cells developed leukemia. These results indicate that WT1 is not necessary for leukemia development in two leukemia models examined. Collectively, WT1 is expressed on immature leukemic cells but not normal HSCs, highlighting its potential as a specific target for therapy. However, the exact function of WT1 in leukemogenesis remains unclear from these studies.

Children are not little adults: just ask their hematopoietic stem cells

HSCs differ during ontogeny in some important parameters, including anatomic site of residence and cell cycling characteristics. In this issue of the JCI, Bowie et al. show that postnatal HSCs as well as fetal liver HSCs in mice are active in the cell cycle at much higher rates than that of adult HSCs; however, this increased frequency of cycling abruptly ceases 4 weeks after birth (see the related article beginning on page 2808). The cycling postnatal HSCs expressed high levels of CXC chemokine ligand 12 (CXCL12, also known as stromal cell-derived factor 1 [SDF-1]), a chemokine previously implicated in stem cell trafficking to the marrow cavity and shown to be expressed by cells within the hematopoietic microenvironment. These cells also possessed an engraftment defect impeding reconstitution in irradiated recipient mice, which was reversible by pretransplant administration of antagonists of the CXCL12 receptor, CXCR4. Such agents are currently clinically available, suggesting that this approach could be used to improve stem cell transplantation and engraftment.

23 Temporal Expression of TLR2 On Hematopoietic Stem Cells in Murine Fetal Liver.

Recently Toll like receptors (TLRs) have been shown to play an essential role in innate immunity, recognizing a specific component of foreign pathogens. TLR2 recognizes gram-positive bacterial cell wall molecules such as peptidoglycan (PG), whereas TLR4 recognizes gram-negative bacterial lipopolysaccharide. TLRs are expressed on various immunological cells such as neutrophils, monocyte/macrophages, dendritic cells and B cells, however, their expression pattern during hematopoietic development/differentiation is not well known. We examined expression of TLR2 or 4 on hematopoietic stem cells (HSCs) in fetal liver. Fetal liver cells at 12.5-16.5 days postcoitum (dpc) were collected and analyzed by flow cytometry. TLR2 and 4 were detected in the murine CD34+c-kit+Sca-1+lineage- (34KSL) population of the12.5 d.p.c fetal liver. 34KSL cells were separated into 3 fractions depending on the expression of TLR2 and 4; TLR2brightTLR4+ cells (7.3%), TLR2dimTLR4- cells (84.3%), and TLR2-TLR4- cells (3.5%). TLR2dimTLR4- and TLR2-TLR4- cells had high colony-forming ability but TLR2brightTLR4+ cells formed very few colonies. In 14.5dpc fetal liver, 90% of 34KSL cells were TLR2dim, however, in 16.5dpc fetal liver, most of the 34KSL cells were TLR2 negative. Interestingly, this TLR2 expression on fetal liver HSCs was synchronized with the expression level of Mac1. When TLR2 bright, TLR2 dim or TLR2- 34KSL cells of the 12.5 dpc fetal liver from Ly5.1 C57BL/6 mice were transplanted into lethally irradiated adult Ly5.2 C57BL/6 mice respectively, TLR2dim 34KSL cells were confirmed to reconstitute the recipient hematopoietic system. To examine the function of TLR2 on fetal liver HSCs, TLR2dim34KSL cells were cultured with/without PG in the presence of SCF and TPO. After 7 days of incubation, cells were collected and analyzed by colony assay and surface markers. Colony forming ability was maintained in both groups but cells differentiated into different lineages. We conclude that TLR2 is temporary expressed on fetal liver HSCs but the role of TLR2 in hematopoiesis is unclear.

Enhanced purification of fetal liver hematopoietic stem cells using SLAM family receptors

Although adult mouse hematopoietic stem cells (HSCs) have been purified to near homogeneity, it remains impossible to achieve this with fetal HSCs. Adult HSC purity recently has been enhanced using the SLAM family receptors CD150, CD244, and CD48. These markers are expressed at different stages of the hematopoiesis hierarchy, making it possible to highly purify adult HSCs as CD150(+)CD48(-)CD244(-) cells. We found that SLAM family receptors exhibited a similar expression pattern in fetal liver. Fetal liver HSCs were CD150(+)CD48(-)CD244(-), and the vast majority of colony-forming progenitors were CD48(+)CD244(-)CD150(-) or CD48(+)CD244(+)CD150(-), just as in adult bone marrow. SLAM family markers enhanced the purification of fetal liver HSCs. Whereas 1 (11%) of every 8.9 Thy(low)Sca-1(+)lineage(-)Mac-1(+) fetal liver cells gave long-term multilineage reconstitution in irradiated mice, 1 (18%) of every 5.7 CD150(+)CD48(-)CD41(-) cells and 1 (37%) of every 2.7 CD150(+)CD48(-)Sca-1(+)lineage(-)Mac-1(+) fetal liver cells gave long-term multilineage reconstitution. These data emphasize the robustness with which SLAM family markers distinguish progenitors at different stages of the hematopoiesis hierarchy and enhance the purification of definitive HSCs from diverse contexts. Nonetheless, CD150, CD244, and CD48 are not pan-stem cell markers, as they were not detectably expressed by stem cells in the fetal or adult nervous system.

Genetic reduction of class IA PI-3 kinase activity alters fetal hematopoiesis and competitive repopulating ability of hematopoietic stem cells in vivo

Class I(A) phosphatidylinositol-3 kinase (PI-3K) is a lipid kinase, which is activated in blood cells by hematopoietic growth factors. In vitro experiments using chemical inhibitors of PI-3K suggest that this kinase is potentially important for hematopoietic stem and progenitor cell (HSC/P) function, and recent studies identify PI-3K as a therapeutic target in treating different leukemias and lymphomas. However, the role of PI-3K in regulating fetal liver or adult hematopoiesis in vivo is unknown. Therefore, we examined PI-3K-deficient embryos generated by a targeted deletion of the p85alpha and p85beta regulatory subunits of PI-3K (p85alpha-/-p85beta+/-). The absolute frequency and number of hematopoietic progenitor cells were reduced in p85alpha-/- p85beta+/- fetal livers compared with wild-type (WT) controls. Further, p85alpha-/-p85beta+/- fetal liver hematopoietic stem cells (HSCs) had decreased multilineage repopulating ability in vivo compared with WT controls in competitive repopulation assays. Finally, purified p85alpha-/-p85beta+/- c-kit+ cells had a decrease in proliferation in response to kit ligand (kitL), a growth factor important for controlling HSC function in vivo. Collectively, these data identify PI-3K as an important regulator of HSC function and potential therapeutic target in treating leukemic stem cells.

Integrin alpha 6 Is Essential for Fetal Hematopoietic Progenitor, but Not Fetal Stem Cell Homing to Bone Marrow.

Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood , 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.

Hematopoietic Defects in Cited2 Mutant Fetal Liver.

Cited2 [cAMP-responsive element-binding protein (CBP)/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2] is a newly identified transcriptional modulator. Knockout of Cited2 gene is embryonic lethal because of heart and neural tube defects. Cited2 binds directly to CBP and p300, which have been shown to be crucial for hematopoietic stem cell self-renewal and proper hematopoietic differentiation, respectively. Cited2 also induces the expression of a polycomb-group gene, Bmi-1, which is essential for self-renewal of adult hematopoietic stem cells. These connections provided rationale to study the potential role of Cited2 in hematopoiesis. Mouse fetal liver is the major hematopoietic organ from day 10 postcoitus until right before birth. The smaller sized Cited2−/− fetal liver and significantly decreased fetal liver cellularity strongly suggest the potential defect in hematopoiesis. In vitro colony formation assay in methycellulose-based medium was used to characterize the hematopoietic progenitors. We found that fetal liver cells from E13.5, 14.5 and E15.5 Cited2−/− embryos gave rise to much less colonies, which reflects the decreased number and proliferative ability of hematopoietic progenitors due to Cited2 deficiency. Immunostaining of lineage-specific cell surface markers followed by flow cytometry was performed to characterize different hematopoietic populations in E14.5 and E15.5 fetal liver of wild type and Cited2−/− embryos. Cited2−/− fetal liver cells displayed a significant reduction in numbers throughout the hematopoietic hierarchy including hematopoietic stem cells (Lin− c-Kit+ Sca-1+), progenitor cells (Lin− c-Kit+), and differentiated cells of different lineages (CD45+, Ter119+, Mac-1+, Gr-1+), thus revealing a multi-level hematopoietic deficiency of Cited2−/− embryos. Long-term reconstitution experiment was then carried out to measure the ability of hematopoietic stem cells from Cited2−/− fetal liver cells to engraft and reconstitute hematopoietic system of congenic recipient mice. Mice transplanted with Cited2−/− fetal liver cells showed reconstitution of T cells whereas a 2-fold decrease in the reconstitution of B cell and myeloid lineages was observed, indicating a compromised ability of Cited2−/− fetal liver hematopoietic stem cells to maintain hematopoiesis. The results suggest an important role of Cited2 in hematopoietic differentiation and a selective function of Cited2 in B lymphoid &myeloid induction. The underlying mechanisms responsible for these defects will be pursued by microarray analysis of gene expression profile of Cited2−/− fetal liver cells, followed by more detailed phenotypic analyses of B and myeloid lineage markers plus in vitro and in vivo functional assays.

D-4F Restores eNOS-Dependent Vasodilation in Sickle Cell Disease by Decreasing Proinflammatory HDL.

Background: Vasoregulation is impaired in human and murine sickle cell disease (SCD). Chronic inflammation and oxidative stress impair vasodilation. High-density lipoprotein (HDL) plays an important role in attenuating inflammatory responses. Previously we showed 4F, an apoA-I mimetic designed to improve HDL function, dramatically restores vasodilation in SCD mice. Here, we examine mechanisms by which D-4F restores vasodilation in SCD mice and in mice made to develop SCD via fetal liver hematopoietic stem cell transplantation (HSCT). Effects of proinflammatory lipids and D-4F were determined in HSCT-SCD- LDL receptor null (Ldlr−/−) mice fed either chow or western diet (WD). The role of HDL was examined in HSCT-SCD-apoA-I null (apoA-I−/−) mice. Finally, the role of eNOS was examined in HSCT-SCD-eNOS deficient (eNOS−/−) mice. Mice were treated with or without D-4F (1mg/kg/d for 6–8 wks).Results: Total cholesterol concentrations in HSCT-SCD-Ldlr−/− mice fed lab chow were slightly increased compared to transgenic SCD mice (40–60 vs. 90–130 mg/dL, p<0.05) with no change in HDL. Acetylcholine-mediated vasodilation (Ach, 10-7 to 10-4M) in HSCT-SCD-Ldlr−/− mice was impaired compared to untreated non-SCD Ldlr−/− mice (10 vs 43%, p<0.05). D-4F restored eNOS-dependent vasodilation in HSCT-SCD-Ldlr−/− mice to the level in non-SCD Ldlr−/− mice. D-4F did not alter total cholesterol or HDL in HSCT-SCD-Ldlr−/− mice but did decrease proinflammatory HDL (580 vs 380, p<0.05), an index of oxidizability. In contrast to HSCT-SCD-Ldlr−/− mice fed chow diet, HSCT-SCD-Ldlr−/− mice fed WD had little to no ACh vasodilation (0–3%). D-4F increased vasodilation slightly in HSCT-SCD-Ldlr−/− fed WD (~12%). Total cholesterol and HDL increased in response to WD in HSCT-SCD-Ldlr−/− mice (p<0.01). D-4F induced minimal changes in total cholesterol, HDL or proinflammatory HDL in these mice. To examine the role of HDL, we found that vasodilation in HSCT-SCD-apoA-I−/− mice was reduced to ~25% compared to 65% in C57BL/6 mice (p<0.01). D-4F nearly doubled vasodilation to ~43% in HSCT-SCD-apoA-I−/− mice (p<0.05). L-NAME (100μM) blocked vasodilation in all HSCT-SCD-apoA-I−/− mice, indicating vasodilation was mediated exclusively by eNOS. In contrast, when we examined the effect of eNOS deficiency, ACh induced minimal increases in vasodilation (~22%). Dissection of cellular mechanisms mediating vasodilation revealed that a small portion HSCT-SCD-eNOS−/− mice was inhibited by L-NAME (i.e., NOS, ~12%), with none mediated by COX-prostacyclin (0%) and a small portion mediated by cytochrome P450 (~10%). Inhibitor studies revealed D-4F restored vasodilation in HSCT-SCD-eNOS−/− mice to ~52% (p<0.05) by predominately a L-NAME-inhibitable mechanism (NOS = 40%; COX-prostacylcin = 0% and cytochrome P450 = 11%).Conclusions: D-4F improves eNOS-dependent vasodilation even when hypercholesterolemia is superimposed on SCD. Measurements of proinflammatory HDL reveal D-4F restores vasodilation by protecting HDL against oxidation. Interestingly, D-4F protects vasodilation even in mice that have low levels of apoA-I-deficient HDL. Taken together, these data indicate proinflammatory HDL plays a critical role in mechanisms by which SCD impairs eNOS-dependent vasodilation and D-4F increases vasodilation, at least in part, by decreasing proinflammatory HDL in SCD.

The Role of Truncated c-kit Receptor (tr-kit) in Maintenance and Differentiation of Hematopoietic Stem Cells and Multipotent Hematopoietic Progenitors.

Intrinsic mechanisms that regulate self-renewal of mammalian stem cells are slowly being elucidated. Self-renewal of stem cells in Drosophila and C. elegans is regulated by members of the conserved Pumilio family of RNA-binding proteins. Previously, we have cloned and characterized two mouse and human Pumilio genes (Pum1 and Pum2), which are abundantly transcribed in hematopoietic stem cells (HSC). To study the function of mammalian Pum proteins in HSC and multipotent progenitors, the RNA-binding domain of Pum2 was over-expressed in a stem cell factor (SCF)-dependent HSC-like cell line EML. In the presence of SCF EML cells undergo SCF-dependent self-renewal and remain undifferentiated. In the presence of various cytokines (IL-3, GM-CSF, G-CSF, Epo, Tpo, IL-7, Flt3L) EML cells differentiate into erythroid, granulocytic, megakaryocytic and lymphoid cell lineages in vitro. The over-expression of Pum2-RBD leads to SCF-independent maintenance of EML cells, and suppresses their mutilineage differentiation in the absence of SCF. This uncoupling of the maintenance and differentiation signals in EML cells is accompanied by (a) an increased expression of the full-length c-kit and a novel truncated c-kit receptor called tr-kit, (b) cell intrinsic, SCF-independent activation of c-kit, and (c) constitutive activation of MAPK, PI3K and PLCγ signaling pathways in the absence of SCF. These results indicate that Pum2 could be supporting maintenance of multipotent hematopoietic cells through regulation of SCF/c-kit signaling pathway. An in depth analysis of the pattern of tr-kit expression in murine fetal liver and bone marrow-derived HSC, multipotent progenitors, lineage-committed progenitors and immature blood cells has shown that tr-kit expression is restricted to cell populations highly enriched for HSC and multipotent progenitors. This observation and the finding that an increased expression of tr-kit protein correlates with SCF-independent maintenance of EML cells, suggest that tr-kit could play an important role in SCF-independent activation of full-length c-kit receptor, and participate in the regulation of the balance between maintenance (self-renewal) and differentiation of HSC and multipotent progenitors. The fact that Pum2 and tr-kit are co-expressed in bone marrow cells enriched for HSC and early multipotent progenitors (e.g. Lin-Sca-1+c-kit+ cells), but not in later progenitors (e.g. Lin-Sca-1−c-kit− cells), suggests an exciting possibility that HSC and early multipotent progenitors utilize distinct SCF-dependent and SCF-independent c-kit signaling pathways. In contrast, more differentiated progenitors that lack self-renewal ability and do not express tr-kit, utilize only the canonical SCF-induced c-kit signaling. In this hypothetical model, the survival and maintenance of HSC and multipotent hematopoietic progenitors is mediated through SCF-independent c-kit signaling, whereas their differentiation depends on the canonical SCF-induced c-kit signaling. We are currently studying the effects of Pum2 and tr-kit over-expression and attenuation on (a) HSC and progenitor cell maintenance and differentiation, (b) HoxB4 and Notch1 pathways, involved in HSC maintenance and expansion, and (c) maintenance and differentiation of HSC expressing SLAM receptor CD150. Further study of Pum2 and tr-kit function could provide important new insights into the molecular regulation of two critical elements of self-renewal, inhibition of differentiation and induction of proliferation.

Gene Expression Profiling of Hematopoietic Stem and Progenitor Cells in Zebrafish.

The differentiation of hematopoietic stem cells (HSCs) into distinct blood lineages is orchestrated by a tight regulation of critical genes during development. To describe the genetic program during the early steps of blood and blood vessel formation in greater detail, we analyzed the expression profiles of zebrafish cloche mutants (clo), in which blood and endothelial cells are completely abrogated, and of isolated hematopoietic and vascular progenitor cell populations from FAC-sorted transgenic lmo2-GFP zebrafish embryos. Consistent with a defect in hemangioblast differentiation, clo mutants at the 5 somite stage were characterized by a prominent downregulation of transcription factors, such as scl and lmo2. As a novel finding, clo mutants at this stage strongly upregulated muscle-related genes, such as myosin, actin, or creatine kinase. FAC-sorted lmo2-GFP cells from 14 somite stage embryos expressed similar hematopoietic transcription factors including scl, lmo2, and gata1. In addition, socs genes, tgfb, fos and jun related genes, and heat shock proteins were also strongly expressed. Comparison with the expression profile of human and mouse fetal liver HSCs revealed a similar regulation between fish and mammals: Expression of 102/168 genes from lmo2-GFP positive cells were also present in mammalian fetal HSCs, indicating an evolutionary highly conserved genetic program of blood formation. In sum, gene expression profiling of zebrafish mutants and of sorted progenitor cells from transgenic zebrafish represent a powerful and versatile tool to define the genetic regulation of vertebrate hematopoietic development.

Placenta as a site for hematopoietic stem cell development

The discovery of a major hematopoietic stem cell (HSC) pool in mid-gestation mouse placenta has defined the placenta as yet another important anatomical site that participates in HSC development. Placental HSC activity starts in parallel with the AGM region, before HSCs are found in circulation or have colonized the fetal liver. Moreover, placental hematopoietic activity culminates in a rapid expansion of the definitive HSC pool, which occurs during the time when the fetal liver HSC reservoir begins to grow. Furthermore, hematopoietic cells in mid-gestation mouse placenta are not instructed for differentiation along the myeloerythroid lineage, as in the fetal liver. These findings suggest that the placenta provides a supportive niche where the definitive hematopoietic stem cell pool can be temporarily established during development. Future studies are needed to characterize the developmental events that lead to the establishment of placental HSC pool, and to define the microenvironmental signals that support this process. Furthermore, if the stem cell-promoting properties of the placental niche can be harnessed in vitro to support HSC formation, maturation, and/or expansion in culture, these assets may greatly improve hematopoietic stem cell-based therapies in the future.

CD144 (VE-cadherin) is transiently expressed by fetal liver hematopoietic stem cells

AbstractHematopoietic stem cells (HSC54) and endothelial progenitors arise from a common embryonic precursor. However, these populations diverge prior to the onset of definitive hematopoiesis, as HSCs become CD45+ and are thought to lose the expression of endothelial markers. After the onset of definitive hematopoiesis, CD144 (vascular endothelial [VE]–cadherin) has been considered a specific marker of endothelial cells. In contrast, we found that virtually all HSC activity from embryonic day 13.5 (E13.5) fetal liver was CD144+. CD144 expression declined on E16.5 fetal liver HSCs and was absent from adult bone marrow HSCs. This identified a new marker that is differentially expressed between fetal and adult HSCs, and enhanced the purification of HSCs from the E13.5 fetal liver. These results emphasize the close developmental relationship between hematopoietic and endothelial cells, while indicating that CD144 is not a specific marker of endothelial cells during fetal development.

Long-Term Bovine Hematopoietic Engraftment with Clone-Derived Stem Cells

Therapeutic cloning by somatic cell nuclear transfer offers potential for treatment of a wide range of degenerative disease. Nuclear transplantation with neo (r)-marked somatic nuclei from 10-13-year-old cows was used to generate cloned bovine fetuses. Clone fetal liver (FL) hematopoietic stem cells (HSC) were transplanted into two busulfan-treated and one untreated nuclear donor cows. Hematopoiesis was monitored over 13-16 months by in vitro progenitor and HSC assays. Chimerism was demonstrated by PCR in blood, marrow, lymph nodes, and endothelium, peaking at levels of 9-17% in blood granulocytes but at lower levels in lymphocyte subsets (0.1-0.01%). Circulating progenitors showed high levels of chimerism (up to 60% neo (r+)) with persisting fetal features. At sacrifice, the animal that had no pre-transplant myelosupression showed persisting donor cells in blood and lymph nodes, and in marrow 0.25% of progenitor cells and a detectable fraction of stem cells were neo (r+). The fetal HSC showed a 10-fold competition advantage over adult HSC. Cloning generated histocompatible HSC capable of long-term multilineage engraftment in a large animal model.

The ETS family transcription factor PU.1 is necessary for the maintenance of fetal liver hematopoietic stem cells

AbstractPU.1 is a member of the ETS family of transcription factors and is required for the development of multiple hematopoietic lineages. PU.1-/- mice die from hematopoietic failure at about embryonic day 18.5 (e18.5) and show a complete absence of B cells, mature T cells, and macrophages. This phenotype suggests that PU.1 may function at the level of the hematopoietic stem cell (HSC) or a multilineage progenitor. To investigate the role of PU.1 in the regulation of HSCs, PU.1-/- embryos were analyzed at various stages of embryonic development. The absolute number and frequency of HSCs were determined by flow cytometric analysis of c-Kit+Thy-1.1loLin-Sca-1+ (KTLS) cells. We found that KTLS cells were absent or severely reduced in PU.1-/- fetal liver from e12.5 to e15.5. Progenitor cells with a c-Kit+Lin-AA4.1+ and c-Kit+Lin-CD34+ phenotype were also severely reduced. In addition, PU.1-/- fetal liver at e14.5 lacked common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) but retained megakaryocyteerythroid progenitors (MEPs). Consistent with the loss of HSC activity, a 10-fold reduction in erythroid progenitors (mature erythroid burst-forming units [BFUEs]) was observed between e14.5 and e16.5. These data suggest that PU.1 plays an important role in the maintenance or expansion of HSC number in murine fetal liver. (Blood. 2004;104:3894-3900)

4-Hydroxynonenal inhibits cell proliferation and alters differentiation pathways in human fetal liver hematopoietic stem cells

During fetal development, the liver serves as the primary hematopoietic organ in which hematopoietic stem cells (HSC) comprise a large proportion of hepatic cell populations. Because HSC are capable of initiating long-term hematopoiesis, injury to these cells may have ramifications with regard to the etiology of blood-borne diseases. In the current study, we examined the effects of 4-hydroxynonenal (4-HNE), a mutagenic α,β-unsaturated aldehyde that can be produced in utero, on HSC proliferation, differentiation, viability and apoptosis. Exposure of HSC to acute single doses of 4-HNE as low as 1 nM inhibited HSC proliferation. Because 4-HNE rapidly disappears from culture media, a multiple dosing régime was also employed to approximate short-term steady state 4-HNE concentrations relevant to physiological oxidative stress. 4-Hydroxynonenal steady state concentrations as low as 1 μM altered HSC differentiation pathways, but did not affect apoptosis or cause cell death. In contrast, exposure to steady state 5 μM 4-HNE elicited a loss in viability, and increased the rate of apoptosis in total HSC populations. Collectively, our data indicate that cellular levels of 4-HNE associated with a low level of oxidative stress cause a loss of proliferation and viability and alter differentiation pathways in human fetal HSC.

Iron Metabolism in the Mice with Targeted Mutations of the DMT1 Gene.

Divalent metal transporter 1 (DMT1) was shown to be important for iron metabolism through studies of the mk/mk mouse, which carries a spontaneous mutation (G185R) resulting in defective intestinal iron absorption and anemia. To further investigate the importance of this transporter in vivo, we inactivated the DMT1 gene through targeted deletion of transmembrane domains 3, 4 and 5. We obtained mice carrying a universally inactivated DMT1 allele (DMT1−/−) and mice carrying a floxed DMT1 allele that could be selectively inactivated by breeding to mice expressing a Cre recombinase transgene. We crossed the floxed mice to mice expressing Cre under the control of the Villin promoter to inactivate DMT1 exclusively in the intestine. All mutations were made on a 129S6/SvEvTac background. For comparison, we backcrossed the mk mutation onto the same background. We noted that, in this context, the mk/mk phenotype was much less severe.DMT1−/− mice were born with substantial liver iron stores, indicating that fetal DMT1 is not required for placental iron transfer or hepatic iron loading. However, DMT1−/− mice invariably died of anemia by day 7, suggesting that DMT1 is important for erythroid iron acquisition but not absolutely required. Transfusions improved survival. The DMT1−/− phenotype was much more severe than the mk/mk phenotype, and compound heterozygous DMT1-/mk mice survived to adulthood. These results confirm that the mk mutation does not result in total loss of protein function.Intestine-specific inactivation of DMT1 caused no abnormalities at birth, but progressive anemia developed thereafter. At 12 weeks, mice lacking intestinal DMT1 were more anemic than mk/mk mice. To investigate the importance of DMT1 in hematopoietic cells, we transferred fetal liver hematopoietic stem cells (HSC) from DMT1−/− animals to irradiated wild type adult mice. Twelve weeks after transfer, DMT1−/− HSC recipients were anemic, but less so than singly transfused DMT1−/− mice or mice lacking intestinal DMT1. DMT1−/− HSC recipients accumulated more liver iron than wild type HSC recipients, suggesting a compensatory increase in intestinal iron absorption and/or altered iron distribution.Surprisingly, though DMT1−/− mice died as neonates, heterozygosity for a null Hfe mutation allowed some DMT1−/−;Hfe+/−animals to survive without transfusions. The effect was more pronounced in animals homozygous for Hfe null mutations. We took advantage of this to compare untransfused DMT1−/− mice with mk/mk mice on an Hfe−/− background. Liver iron content and hemoglobin levels of both mk/mk;Hfe−/− and DMT1−/−;Hfe−/− mice were significantly lower than DMT1+/+;Hfe−/− mice, as expected. Again, mk/mk mice were less severely affected than DMT1−/− mice.We have confirmed that DMT1 plays primary roles in intestinal iron absorption and erythroid iron acquisition but it is likely that there are minor, alternative iron uptake mechanisms. The mk mutation allows for considerable residual iron transport activity. Inactivation of Hfe ameliorates the effects of DMT1 deficiency through an as yet unknown mechanism.

Tissue Factor Deficiency Decreases Sickle Cell-Induced Vascular Stasis in a Hematopoietic Stem Cell Transplant Model of Murine Sickle Cell Disease.

There is considerable evidence for increased levels of procoagulant proteins, thrombin generation and tissue factor activity in patients with sickle cell disease (SCD). Both thrombin and tissue factor have potent proinflammatory effects. We found increased fibrin deposition in organs that develop SCD vaso-occlusive pathologies in the Berkeley murine model of SCD. In addition, we demonstrated that transplantation of sickle hematopoietic stem cells (HSC) into mice deficient in fibrinogen (SCD-Fib−/− mice) resulted in a decreased number and size of liver infarcts. These data suggest that fibrin clot formation is critical for the final irreversible stoppage of blood flow that can progress to focal tissue infarction. However, the SCD-Fib−/− mice still develop other typical organ pathologies associated with SCD, including focal sites of prominent vascular congestion that are likely due to persistent endothelial injury and inflammation. Because patients with afibrinogenemia have high levels of circulating thrombin, the persistent organ pathology in the SCD-Fib−/− mice may also be linked to the increased exposure to thrombin's potent proinflammatory effects. In agreement, we now have evidence for increased circulating thrombin-antithrombin (TAT) complexes in SCD-Fib−/− mice compared to SCD-wt transplant control mice (54 ± 25 vs 9.9 ± 3.5 ng/mL, p=0.04) demonstrating increased thrombin generation in the SCD-Fib−/− mice. In addition, soluble VCAM-1, a marker of endothelia injury, was increased in SCD-Fib−/−mice compared to SCD-wt mice (843 ± 90 vs 628 ± 110). In order to determine whether the tissue factor pathway contributes significantly to proinflammatory pathologies observed in SCD, we transplanted fetal liver HSC from mice that predominately express human sickle hemoglobin (mα−/−, mβ−/−, Tg[HbS]+: “SCD mice” or hemizygous mα−/−, mβ+/−, Tg[HbS]x2: “hSCD2 mice,” Berkeley model) into mice deficient in tissue factor (mTF−/−, hTF+: “TFLow mice” that express ~1%TF levels) or control heterozygote mice (mTF+/−, hTF+) following lethal irradiation. Four months following transplant, we obtained blood samples for engraftment and hematologic studies (>90% engraftment required for inclusion in this study), followed by perfusion of anesthetized animals. Perfused organs were divided with one part fixed for histologic study. Frozen nonfixed sections of the organs were homogenized and organ Hb content determined per gram organ weight. We found that tissue factor deficiency (SCD-TFLow and hSCD2-TFLow mice) resulted in a dramatic reduction of vascular congestion in liver histologic sections that was confirmed by quantitation of organ-specific trapped Hb: 1.2 ± 0.3 vs 6.3 ± 1.7 mg Hb/gm liver in hSCD2-TFLow vs hSCD2-TF+/− control mice (n = 8–9 per group, p<0.001) with similar results in a smaller number of SCD-TFLow mice. These data suggest that the tissue factor pathway contributes importantly to vascular inflammation and cellular stasis. Thus, while the coagulation pathway appears to be a critical component of HbS-induced tissue infarction, endothelial injury and inflammation also significantly contribute to HbS-induced organ pathology. These data also suggest that targeted anticoagulation therapies may ameliorate the focal organ infarction and/or vascular inflammation that are prevalent in SCD.

ABC transporter activities of murine hematopoietic stem cells vary according to their developmental and activation status

AbstractPrimitive hematopoietic cells from several species are known to efflux both Hoechst 33342 and Rhodamine-123. We now show that murine hematopoietic stem cells (HSCs) defined by long-term multilineage repopulation assays efflux both dyes variably according to their developmental or activation status. In day 14.5 murine fetal liver, very few HSCs efflux Hoechst 33342 efficiently, and they are thus not detected as “side population” (SP) cells. HSCs in mouse fetal liver also fail to efflux Rhodamine-123. Both of these features are retained by most of the HSCs present until 4 weeks after birth but are reversed by 8 weeks of age or after a new HSC population is regenerated in adult mice that receive transplants with murine fetal liver cells. Activation of adult HSCs in vivo following 5-fluorouracil treatment, or in vitro with cytokines, induces variable losses in Rhodamine-123 and Hoechst 33342 efflux activities, and HSCs from mdr-1a/1b-/- mice show a dramatic decrease in Rhodamine-123 efflux ability. Thus, the Rhodamine-123 and Hoechst 33342 efflux properties of murine HSCs fluctuate in the same fashion as a number of other HSC markers, suggesting these are regulated by a common control mechanism that operates independently of that regulating the regenerative function of HSCs. (Blood. 2004;103:4487-4495)