Mouse Fetal Liver Research Articles

Expression of IL-5Rα on B-1 cell progenitors in mouse fetal liver and involvement of Bruton's tyrosine kinase in their development

B-1 cells are a subset of B cells responsible for the production of natural antibodies. Although the amount of natural antibody is tightly regulated, how this regulation occurs remains unknown. We examined the expression of IL-5 receptor, a cytokine receptor critical for homeostatic proliferation of B-1 cells, on B-1 cell progenitors in the fetal liver. We identified B-1 progenitors expressing low levels of IL-5 receptor α chain (IL-5Rα) and eosinophil progenitors expressing higher levels of IL-5Rα in the fetal liver. Moreover, the number of these B-1 progenitors were significantly reduced in the fetuses of mice deficient in Bruton's tyrosine kinase (Btk), even though IL-5 and thymic stroma lymphopoietin signaling are intact in early B lineage cells in Btk-deficient mice. These data suggest that IL-5 is possibly involved in B-1 cell development and an uncharacterized, Btk-dependent regulatory signaling pathway is involved in unexpectedly early stages of B-1 cell differentiation.

MicroRNA‐23b cluster microRNAs regulate transforming growth factor‐beta/bone morphogenetic protein signaling and liver stem cell differentiation by targeting Smads†

Transforming growth factor-beta / bone morphogenetic protein (TGFbeta/BMP) signaling has a gradient of effects on cell fate choice in the fetal mouse liver. The molecular mechanism to understand why adjacent cells develop into bile ducts or grow actively as hepatocytes in the ubiquitous presence of both TGFbeta ligands and receptors has been unknown. We hypothesized that microRNAs (miRNAs) might play a role in cell fate decisions in the liver. miRNA profiling during late fetal development in the mouse identified miR-23b cluster miRNAs comprising miR-23b, miR-27b, and miR-24-1 and miR-10a, miR-26a, and miR-30a as up-regulated. In situ hybridization of fetal liver at embryonic day 17.5 of gestation revealed miR-23b cluster expression only in fetal hepatocytes. A complementary (c)DNA microarray approach was used to identify genes with a reciprocal expression pattern to that of miR-23b cluster miRNAs. This approach identified Smads (mothers against decapentaplegic homolog), the key TGFbeta signaling molecules, as putative miR-23b cluster targets. Bioinformatic analysis identified multiple candidate target sites in the 3' UTRs (untranslated regions) of Smads 3, 4, and 5. Dual luciferase reporter assays confirmed down-regulation of constructs containing Smad 3, 4, or 5, 3' UTRs by a mixture of miR-23b cluster mimics. Knockdown of miR-23b miRNAs during hepatocytic differentiation of a fetal liver stem cell line, HBC-3, promoted expression of bile duct genes, in addition to Smads, in these cells. In contrast, ectopic expression of miR-23b mimics during bile duct differentiation of HBC-3 cells blocked the process. Our data provide a model in which miR-23b miRNAs repress bile duct gene expression in fetal hepatocytes while promoting their growth by down-regulating Smads and consequently TGFbeta signaling. Concomitantly, low levels of the miR-23b miRNAs are needed in cholangiocytes to allow TGFbeta signaling and bile duct formation.

Y-Box Binding Protein-1 Down-Regulates Expression of Carbamoyl Phosphate Synthetase-I by Suppressing CCAAT Enhancer-Binding Protein-Alpha Function in Mice

Carbamoyl phosphate synthetase-I (CPS1) is a key enzyme in the urea cycle and patients with defects in the function or expression of CPS1 suffer from hyperammonemia. CPS1 is expressed in the liver at neonatal and adult stages in a CCAAT enhancer-binding protein-alpha (C/EBPalpha)-dependent manner. Despite expression of C/EBPalpha, CPS1 is not expressed in fetal liver, indicating an additional factor is involved in the regulation of CPS1 expression. The aim of this study was to elucidate the mechanism of CPS1 expression. Microarray was performed to find Y-box binding protein-1 (YB-1) that was expressed in mouse fetal liver. The role of YB-1 in CPS1 expression was investigated by overexpression of YB-1 in mouse fetal liver culture and luciferase reporter assays using the CPS1 promoter. Chromatin immunoprecipitation assay was used to examine recruitment of YB-1 to the CPS1 promoter in vivo. Expression of YB-1 and CPS1 was inversely correlated in vivo, and YB-1 inhibited CPS1 expression and ammonia clearance in fetal liver culture. Although YB-1 was not expressed in adult liver, acute liver injury up-regulated YB-1 and down-regulated CPS1, accompanying an increase of the serum ammonia level. YB-1 inhibited C/EBPalpha-induced transcription from the CPS1 promoter via the Y-box near the C/EBPalpha-binding site. Chromatin immunoprecipitation assays demonstrated that YB-1 was recruited to the CPS1 promoter in fetal and injured adult liver, but not in normal adult liver. YB-1 is a key regulator of ammonia detoxification by negatively regulating CPS1 expression via suppression of C/EBPalpha function.

Interleukin-1 regulates hematopoietic progenitor and stem cells in the midgestation mouse fetal liver

Hematopoietic progenitors are generated in the yolk sac and aorta-gonad-mesonephros region during early mouse development. At embryonic day 10.5 the first hematopoietic stem cells emerge in the aorta-gonad-mesonephros. Subsequently, hematopoietic stem cells and progenitors are found in the fetal liver. The fetal liver is a potent hematopoietic site, playing an important role in the expansion and differentiation of hematopoietic progenitors and hematopoietic stem cells. However, little is known concerning the regulation of fetal liver hematopoietic stem cells. In particular, the role of cytokines such as interleukin-1 in the regulation of hematopoietic stem cells in the embryo has been largely unexplored. Recently, we observed that the adult pro-inflammatory cytokine interleukin-1 is involved in regulating aorta-gonad-mesonephros hematopoietic progenitor and hematopoietic stem cell activity. Therefore, we set out to investigate whether interleukin-1 also plays a role in regulating fetal liver progenitor cells and hematopoietic stem cells. We examined the interleukin-1 ligand and receptor expression pattern in the fetal liver. The effects of interleukin-1 on hematopoietic progenitor cells and hematopoietic stem cells were studied by FACS and transplantation analyses of fetal liver explants, and in vivo effects on hematopoietic stem cell and progenitors were studied in Il1r1(-/-) embryos. We show that fetal liver hematopoietic progenitor cells express the IL-1RI and that interleukin-1 increases fetal liver hematopoiesis, progenitor cell activity and promotes hematopoietic cell survival. Moreover, we show that in Il1r1(-/-) embryos, hematopoietic stem cell activity is impaired and myeloid progenitor activity is increased. The IL-1 ligand and receptor are expressed in the midgestation liver and act in the physiological regulation of fetal liver hematopoietic progenitor cells and hematopoietic stem cells.

A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver

Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or α-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.

Gene expression profiling in the liver and lung of perfluorooctane sulfonate-exposed mouse fetuses: Comparison to changes induced by exposure to perfluorooctanoic acid

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are environmental contaminants found in the tissues of humans and wildlife. They are activators of peroxisome proliferator-activated receptor-alpha (PPARα) and exhibit hepatocarcinogenic potential in rats. PFOS and PFOA are also developmental toxicants in rodents and PFOS has been shown to induce pulmonary deficits in rat offspring. Pregnant CD-1 mice were dosed with 0, 5, or 10mg/kg PFOS from gestation days 1–17. Transcript profiling was conducted on the fetal liver and lung. Results were contrasted to data derived from a previous PFOA study. PFOS-dependent changes were primarily related to activation of PPARα. No remarkable differences were found between PFOS and PFOA. Given that PPARα signaling is required for neonatal mortality in PFOA-treated mice but not those exposed to PFOS, the neonatal mortality observed for PFOS may reflect functional deficits related to the physical properties of the chemical rather than to transcript alterations.

Inactivation of FIP200 Leads to Inflammatory Skin Disorder, but Not Tumorigenesis, in Conditional Knock-out Mouse Models

FIP200 (focal adhesion kinase family interacting protein of 200 kDa) has been shown to interact with other proteins to regulate several intracellular signaling pathways. To study a potential role of FIP200 in tumorigenesis and possibly other disease processes in vivo, we created and analyzed murine mammary tumor virus-Cre-mediated FIP200 conditional knock-out (CKO) mice. We found that deletion of FIP200 in mammary epithelial cells did not result in spontaneous development of breast cancer. Moreover, deletion of FIP200 did not further accelerate or inhibit lymphomagenesis induced by inactivation of p53 in mice. Interestingly, however, FIP200 and p53 double conditional knock-out (dCKO) mice exhibited significant hyperplasia of epidermis (acanthosis), thickening of the cornified layer (hyperkeratosis), and increased vascularity in the dermis. FIP200 CKO mice also showed similar, although less severe, skin defects as dCKO mice. Analyses of primary keratinocytes isolated from dCKO mice did not detect increased proliferation of these cells in vitro, suggesting that epidermis hyperproliferation is not epidermal cell-autonomous but may be a consequence of increased inflammation triggered by immune cells in vivo. Consistent with this possibility, we found infiltration of leukocytes including T cells, macrophages, and granulocytes into the dermis and epidermis, associated with activation of NF-kappaB and increased expression of several proinflammatory cytokines and chemokines in skin of the dCKO mice. We further found that cultured FIP200 KO keratinocytes showed reduced NF-kappaB phosphorylation in response to tumor necrosis factor alpha stimulation, suggesting a paracrine regulation of aberrant NF-kappaB activation in the skin microenviroment of dCKO and FIP200 CKO mice. Together, these results demonstrate that ablation of FIP200, although not promoting tumorigenesis, can lead to skin inflammatory disorders, suggesting a novel function of FIP200 in the maintenance of normal skin homeostasis in vivo.

Spheroid array of fetal mouse liver cells constructed on a PEG-gel micropatterned surface: upregulation of hepatic functions by co-culture with nonparenchymal liver cells

A spheroid array of fetal mouse liver cells, which comprise various immature cells, was constructed on a PEG-gel micropatterned surface and its hepatic activity and degree of differentiation induction were significantly upregulated by co-culture with nonparenchymal liver cells as feeder-cells.

TLX1 (HOX11) Immortalization of Embryonic Stem Cell–Derived and Primary Murine Hematopoietic Progenitors

The ability to generate genetically engineered cell lines is of great experimental value. They provide a renewable source of material that may be suitable for biochemical analyses, chromatin immunoprecipitation assays, structure-function studies, gene function assignment, and transcription factor target gene identification. This unit describes protocols for TLX1 (HOX11)-mediated immortalization of murine hematopoietic progenitors derived from in vitro differentiated murine embryonic stem cells, or from primary mouse fetal liver or bone marrow. A wide variety of hematopoietic cell types have been immortalized using these procedures including erythroid, megakaryocytic, monocytic, myelocytic, and multipotential cell types. These lines are typically cytokine dependent for their survival and growth.

Induction of Hyperproliferative Fetal Megakaryopoiesis by An N-Terminally Truncated GATA1 Mutant.

Two types of GATA1-related leukemias have been noticed. One is the erythroid leukemia develops in mice as a consequence of deficient expression of GATA1, while the other is acute megakaryoblastic leukemia (AMKL) that arises in Down syndrome (DS) children as a consequence of somatic N-terminally truncation (ΔNT) mutation of GATA1 (reviewed in Shimizu et al, Nat Rev Cancer 2008). We hypothesized that modeling of the human mutation in mice might provide novel insights into human TMD and DS-AMKL, since the GATA1 N-terminal (NT) function is apparently important in these disorders. A previous report showed that immature megakaryocytic progenitors in the yolk sac and early fetal liver of knock-in mice that expressed a mutant GATA1 protein lacking amino acids 3 to 63 (and not 1–83, as in the DS-associated AKML patients) developed a hyperproliferative phenotype, but also that these aberrant cells disappeared from fetal blood by E14.5. The same phenotype was detected in conditional knockout mice in which Gata1 exon 2 (which encodes the wild-type start codon) was deleted. Since this phenotype was substantially different from human TMD, the contribution of the GATA1 NT 83 amino acids to human TMD pathogenesis remained obscure. Therefore, we adopted a transgenic complementation/rescue approach to more directly analyze NT domain function. We generated mice that were rescued from GATA1-deficient lethality by transgenic expression of a GATA1 mutant lacking the N-terminal 83 amino acids, a protein that is identical to the mutant GATA1 factor encountered in human TMD and DS-AKML cases. We discovered that expression of this shortened GATA1 protein in mice phenocopies human TMD in DS children preceding AMKL; immature megakaryocytes accumulate massively in the perinatal livers of mutant mice. However, unlike the usual disease progression in humans, these mouse mutants never develop leukemia. These results unequivocally revealed that expression of the short form of GATA1 is sufficient to induce hyperproliferative fetal megakaryopoiesis, but additional mutation(s) to the GATA1/Gata1 N-terminal deletion is necessary to transform those hyperproliferative megakaryocytic cells into overt leukemic cells in both mouse and human. Thus, this model significantly clarifies our understanding of the distinct molecular mechanisms by which the GATA1 protein can contribute to the multi-step leukemogenesis.

Nucleolar Stress and p53 Mediated Erythroid Failure in Diamond Blackfan Anemia

The discovery that several ribosomal protein genes can be mutated in DBA suggests that ribosomal protein gene mutations may account for many or all cases of DBA, and focuses attention on the ribosome. While experiments in yeast and mammalian cells show that RPS19 depletion or mutation leads to a block in ribosomal RNA biosynthesis, this result does not explain why erythropoiesis is so severely affected in DBA. We hypothesize that during fetal development immature erythroid cells proliferate more rapidly than other lineages and therefore require very high ribosome synthetic rates to generate sufficient capacity for translation of erythroid specific transcripts that must take place before these unique cells enucleate. To test this kinetic hypothesis we measured RNA biogenesis in primary mouse fetal liver cells and reported previously that during the first 24 hours cell number increases 3–4 fold while, remarkably, there is a 6-fold increase in RNA content during the same period, suggesting that the cells accumulate an excess of ribosomal RNA (80% of measured RNA) during early erythropoiesis. Retrovirus infected siRNA RPS19 knockdown cells show reduced proliferation of FACS sorted GFP positive cells at 48 hours. Although the cell yield is reduced, the differentiation pattern of the surviving GFP positive cells is similar to that of the controls. While quantitative RT-PCR analysis shows that RPS19 mRNA is rapidly depleted, Western analysis during this time course does not show a deficiency of RPS19 protein. This suggests strongly that the proliferative defect is not due to insufficiency of RPS19 protein, and is more likely due nucleolar stress induced by the block in ribosome biogenesis. Molecular consequences could lead to redistribution of cell cycle proteins normally resident in the nucleolus with consequent p53 mediated cell cycle arrest and or apoptosis. To test this hypothesis we used a culture system that allows expansion without differentiation of immature cells in SCF, EPO, IGF-1 and dexamethasone. Under these conditions proliferation of siRNA expressing precursors is reduced with an increased proportion arrested in G0/G1 in the knockdown cells. Furthermore, p53 is increased in the knockdown cells. Taken together, these data suggest that RPS19 insufficient cells undergo a nucleolar stress response and erythroid cells proliferate poorly because of p53 mediated cell cycle arrest and apoptosis.

Oxidative Stress Signaling Pathway of Heme Regulated eIF2α Kinase in mitigating the Severity of β-Thalassemia

Maturation of erythroid precursors requires active synthesis of hemoglobin which consists of two pairs of α- and β-globin subunits with each monomer bound to a heme moiety. Heme Regulated Inhibitor (HRI) is the only eIF2αkinase responsible for the balanced synthesis of heme and globin at translational level in erythroid cells. Activation of HRI in heme deficiency leads to phosphorylation of the α-subunit of eukaryotic initiation factor (eIF2α) and inhibition of protein synthesis. HRI is also activated by denatured proteins and oxidative stress. In addition to general inhibition of protein synthesis, phosphorylation of eIF2α (eIF2αP) also leads to the induction of a stress signaling pathway. Activating transcription factor 4 (Atf4) mRNA is preferentially translated amidst global inhibition of protein synthesis. Atf4 activates transcription of stress response proteins, Chop (CCAAT/enhancer binding protein homologous protein-10) and the non-enzymatic cofactor of eIF2α phosphatase (PP1A) Gadd34. These stress response proteins help cells in mitigating the stress. While the role of HRI in translational regulation of non-nucleated reticulocytes is well established, the HRIdependent Atf4 stress signaling pathway of nucleated erythroid precursors is unknown.Sodium arsenite toxicity was used as a model system of oxidative stress to elucidate the HRI signaling pathway in Hri +/+ and −/− E14.5 mouse fetal liver erythroid precursors. In HRI deficiency, erythroid precursors were more sensitive to arsenite toxicity with decreased cell viability and increased apoptosis, by caspase 3 executed intrinsic apoptotic pathway. HRI was activated by autophosphorylation as early as 15 minutes following arsenite treatment. In addition to increased eIF2αP, there was induction of Atf4, Chop and Gadd34 in Hri+/+ fetal liver cells. Importantly, in Hri−/− cells neither the phosphorylation of eIF2α nor the expression of Atf4, Chop and Gadd34 was increased upon arsenite treatment. In addition, we also observed HRI dependent induction of Heme Oxygenase 1 (HO-1) that plays a pivotal role in adaptation to oxidative stress. These results demonstrate that HRI induces a signaling pathway for adaptive gene expression to protect the nucleated erythroid precursors from apoptosis upon oxidative stress.Iron overload, accumulation of unpaired α-globin and oxidative stress are well documented in β-thalassemia. Recently, HRI was discovered to be necessary for the survival of β-thalassemic mice. β-thalassemic mice lacking one copy of HRI (Hri+/− Hbb−/−) also manifest a more severe syndrome of the disease. We have investigated the activation of eIF2αP/Atf4 signaling pathway in Hri+/−Hbb−/− β-thalassemic erythroid cells using eIF2αP phosphatase (Gadd34) inhibitor salubrinal. Treatment of reticulocytes from Hri+/−Hbb−/− mice with salubrinal increased eIF2αP and resulted in inhibition of newly synthesized globin protein synthesis. The decreased globin protein synthesis also resulted in decreased aggregation of the unpaired α-globins. Furthermore, treatment of salubrinal in nucleated fetal liver erythroblasts also increased Chop expression and decreased apoptosis. Thus, activation of the eIF2αP/Atf4 pathway by small chemicals might be a novel pharmaceutical approach to decrease proteotoxicity and apoptosis for the treatment of β-thalassemia.

IGH@ Translocations Involving the Pseudoautosomal Region 1 (PAR1) of Both Sex Chromosomes Deregulate the Cytokine Receptor-Like Factor 2 (CRLF2) Gene in B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL)

We have recently described recurrent IGH@ translocations in BCP-ALL, which result in deregulated expression of the translocated oncogene. We carried out screening for novel IGH@ translocations in BCP-ALL by fluorescence in situ hybridization (FISH) and identified two cryptic translocations involving PAR1 of either sex chromosomes in 29 patients: t(X;14)(p22;q32) (n=18) and t(Y;14)(p11;q32) (n=7) (X or Y unknown, n= 4) and 2 BCP-ALL cell lines with t(Y;14)(p11;q32). The median age of the patients was 18 years (range 4–76 years) and median WBC was 70 ×109/l (range 1–342). Follow-up data was available for 16 patients. Among 5 children diagnosed 1996–2001, 1 failed to remit, 1 suffered an early death and 3 relapsed (16m, 54m, and 64m post diagnosis). To date, all 5 children diagnosed 2004–2005 are in first continuing complete remission (CCR) after 33–46 months, suggesting an improved outcome on the current childhood ALL treatment trial. All 6 adults were diagnosed 2004–2006: 2 died within a year (1 in remission and 1 post transplant); 2 relapsed and died within 1 year and 2 are in 1st CCR after 20 and 36 months. These observations indicate a variable outcome among adults. Translocation breakpoint cloning from IGHJ by long distance inverse PCR and subsequent FISH mapping identified CRLF2, also known as thymic stromal-derived lymphopoietin receptor, at Xp22 and Yp11, to be juxtaposed to IGH@ in these translocations. Breakpoints were cloned in 11 patients and clustered between 2–27kb centromeric of the 3′ UTR of CRLF2. Expression levels of CRLF2, measured by qRT-PCR in 4 patients and the cell lines, was 500–6,500 fold higher than the control cohort of BCP-ALL patients without the translocation. This overexpression confirmed CRLF2 to be the target gene. CRLF2 and IL2RGC bind to IL7RA to form functional receptors for TSLP and IL7, respectively. However, neither IL2RGC nor IL7RA were aberrantly expressed. The presence of associated genomic copy number alterations was shown by array-based comparative genomic hybridization and FISH. As described for BCP-ALL in general, the significant changes included deletions of the B cell differentiation genes: PAX5 and IKAROS, as well as the cell cycle control gene, CDKN2A. The cell lines revealed some biochemical insights into the functional consequences of this translocation involving PAR1. Cell surface expression of CRLF2 was demonstrated, together with constitutive tyrosine phosphorylation of JAK2 and STAT5, the signaling intermediates of the classical JAK-STAT pathway. Further evidence of increased STAT5 activation came from retroviral infection of normal mouse fetal liver cells with human CRLF2, which sustained the proliferation of B-cell precursors in vitro. Conversely, initial studies of CRLF2 knockdown in the cell lines using shRNA demonstrated decreased proliferation. Together, these results show for the first time, the involvement of both sex chromosomes in an IGH@ translocation in leukemia, resulting in deregulated expression of CRLF2 and constitutive JAK-STAT activation. These data suggest CRLF2 as a potential therapeutic target in this subset of patients.Laboratories of MJSD, RS and CJH contributed equally to this work.

SIRT1 Deficiency Suppresses the Maintenance of Hematopoietic Stem Cell Pool.

Sir2 (silent information regulator 2) is a member of a gene family (sirtuins) encoding NAD(+)-dependent histone deacetylases, which leads to increased DNA stability and prolonged lifespan in Saccharomyces cerevisiae and Caenorhabditis elegans. In mammalians, SIRT1 has also been found to function as a deacetylase for numerous protein targets involved in various cellular pathways, including stress responses, apoptosis, and neural axonal degeneration. However, the effects of SIRT1 on hematopoiesis remains unknown. We previously reported that the SIRT1 inhibitor, nicotinamide(NA), promoted the differentiation of murine hematopoietic stem/progenitor cells, and its activator, Resveratrol, suppressed the differntiation. In this report, we analysed the differentiation of stem/progenitor cells derived from SIRT1 KO mice. Because SIRT1 KO mice can survive less than a week after birth, we performed experiments using c-Kit(+)Lineage(−)Sca-1(+) cells (KSLs) derived from KO mice fetal liver. At first, we cultured KSLs with the cytokine cocktail containing SCF, IL-6, Flt3L, and TPO, which is utilized for the expansion of stem cells. After five day culture, we estimated the population which remains in KSL fraction. As a result, percentage of KSLs from KO fetal liver was less than 5%, while that from WT was about 15%. We also examined the colony formation of KO and WT fetal liver KSL cells using replating assays. At the first plating, total number of colonies developed from KO fetal liver KSLs was smaller than that from WT by 30–40%, and at the third plating, there could be detected no colonies from KO, while 20–30 colonies were observed from WT. Furthermore, we performed serial transplantation assays using WT and KO fetal liver KSLs. Although after primary transplant, we detected no significant difference in repopulation from KO KSLs compared to WT controls, three weeks after secondary transplant, % chimerism from KO KSLs was reduced to 1/2 compared with that from WT KSLs. These results suggested that Sirt1 suppresses the differentiation and promotes self-renew of hematopoietic stem/progenitor cells. To dissect the roles of target molecules of Sirt1 in suppression of differentiation, we first examined the mRNA expressions of some cell cycle-relating molecules in KO and WT fetal liver KSLs. As consequence, p16Ink4A and p19Arf were detected only in KO KSLs. Then we analyzed the roles of molecules which may effect those expressions. First, we examined the effects of MAPkinases inhibitors on the differentiation of KO and WT fetal liver KSLs. During the culture with SCF, IL-6, Flt3L, and TPO, the addition of p38 inhibitor(SB202190), or MEK1 inhibitor(PD98059), or JNK inhibitor did not change the effects of the SIRT1 targeting. Then it was suggested that MAPkinase pathways have little relation with the SIRT1-induced suppression of differentiation. Next we examined the role of p53, which was reported to combine with SIRT1 and to be deacetylated and repressed by SIRT1. KO and WT fetal liver KSLs were cultured with p53 inhibitor (pifithrin?), which partially cancelled the promotion of differntiation in SIRT1 KO KSLs. This result suggested that SIRT1 might inhibit differentiation of KSLs partially by antagonizing p53 activity. Next we examined the role of Foxo3a, a downstream molecule of SIRT1. Enforced expression of constitutive active form of Foxo3a(FKHRL1TM) also cancelled the promotion of differentiation in SIRT1 KO KSLs. As conclusion, we demonstrate that SIRT1 suppresses the differentiation of hematopoietic stem/progenitor cells by antagonizing p53 and enhancing Foxo3a activities, and contributes to maintenance of stem cell properties and stem cell pool.

A Closer Look at Cellular Iron Metabolism in IRP2 Deficient Erythroblasts.

Developing red blood cells are the major consumers of body iron which is indispensable for the enormous production of heme for hemoglobin synthesis. The uptake of iron occurs via binding of iron-loaded transferrin to its cognate receptor (TfR). Thereafter the iron is shuttled to the mitochondria where it is incorporated into protoporphyrin IX to form heme. Excess iron is enclosed within the iron storage protein ferritin. Coordinated control between iron uptake and storage is mainly achieved by the post-transcriptional regulation of TfR1 and ferritin synthesis by the iron regulatory proteins IRP1 and IRP2. Recently, two groups independently created mice lacking either IRP1 or IRP2 and showed that only IRP2 deficient mice developed microcytic hypochromic anemia. Both groups observed a reduction in TfR1 protein expression levels in the developing red blood cells of IRP2 knockout animals and suggested that the decrease in receptor levels is responsible for the development of anemia.For a more detailed analysis of how the loss of IRP2 expression influences iron metabolism and hemoglobinization during terminal erythroid differentiation, we isolated CFU-E-like erythroid cells from mouse fetal liver of wild type, IRP1 and IRP2 knock out animals. In vitro cultivation of these primary erythroid cells and their synchronous induction for differentiation allowed us to study their cellular iron metabolism at different time points. We analyzed the extent of hemoglobinization and cell size as well as the expression of ferritin and TfR1 during various stages of erythroid differentiation in IRP1, IRP2 and wild type cells. In agreement with the published phenotype of microcytic hypochromic anemia, only erythroblasts lacking IRP2 exhibited a reduction in hemoglobinization and showed a significant increase in ferritin protein levels before and after induction of differentiation. In contrast, TfR1 protein expression levels on the cell surface were significantly decreased in IRP2 deficient cells until 24h of differentiation, but converged with those of wild type cells at 48h of differentiation at the time point at which hemoglobinization is fully in progress. Moreover, measurement of 59Fe uptake and its cellular distribution showed that there is significantly more 59Fe located in cytosolic ferritin of IRP2 knock out cells at all time points compared to their wild type counterpart. In summary, these results suggest that not only the reduced expression of TfR1, but also the up-regulation of ferritin, play important roles in the development of anemic phenotype in IRP2 knock out mice.This work was supported by the Canadian Institutes of Health Research and the Canadian Blood Services.

Differentiation of Mouse Hepatic Progenitor Cells Induced by Hepatocyte Nuclear Factor-4 and Cell Transplantation in Mice With Liver Fibrosis

Hepatocyte nuclear factor-4 (HNF-4) plays a central role in the differentiation process of hepatic cells. We investigated the effects of an overexpression of HNF-4 on hepatic progenitor cells isolated from a fetal mouse liver and transplantation of the cells in a mouse model of liver fibrosis. Hepatic progenitor cells were isolated from the embryonic day 14.0 fetal mouse livers and were purified by magnetic cell sorting to remove the hematopoietic cells. We transfected adenovirus-mediated HNF-4 into the cells, and analyzed the expressions of the liver-specific functions using reverse-transcription polymerase chain reaction and Northern blotting. HNF-4-overexpressing hepatic progenitor cells were then injected into recipient mice, which were treated with dimethylnitrosamine and 30% partial hepatectomy. After 5 days of culture, the cells located in the center of the aggregates were stained positive for albumin, but the peripheral cells for cytokeratin 19. Adenovirus-mediated HNF-4 gene transfer resulted in increases in the expressions of HNF-4, apolipoprotein (Apo)A1, ApoC3, and pregnane X receptor messenger RNA. The mice treated with HNF-4-transfected progenitor cells survived significantly longer than the control mice (P=0.004). The plasma levels of albumin, total cholesterol, and glucose were higher in the mice treated with cells transfected by HNF-4 than in the control mice. These findings demonstrate that adenovirus-mediated HNF-4 transfection induces the differentiation from hepatic progenitor cells to hepatic parenchymal cells in vitro. These cells may be useful as a source for cell transplantation in liver diseases.

In this issue: Proteomics 21/2008

AbstractIn this issue of Proteomics you will find the following highlighted articles:When doing your best is too goodSometimes it pays to go back and question your assumptions, particularly if you are about to embark on a project with a lot of samples to process and analyze and, more particularly, if the instrumentation has changed substantially in the mean time. Villén et al. found a surprise at the end of their reevaluation of phosphopeptide identification. To start, they thought that the availability of 2–D and 3–D ion traps and high accuracy, high sensitivity would speed the process along substantially. Not so. First, neutral losses threatened to swamp out the MS2 spectrum from the 3–D ion trap, then they almost washed out the b– and y–type ions that comprise a critical internal ruler. The overabundance could be attenuated by using a new data dependent neutral loss MS3 method with 3–D ion traps. However, switching to high resolution ion traps reduced spectrum collection speed. . . Compromise time.Villén, J et al., Proteomics 2008, 8, 4444–4452.TGFbeta: a triskadecapoidal growth/ungrowth factor?One thing proteins do much more effectively than nucleic acids is multi–tasking. But they also start with a much larger tool chest – 20 amino acids vs. 4 nucleotides; dozens of post–translational modification possibilities vs. a few methylation sites. . . Transforming growth factor b takes advantage of its flexibility very effectively in regulating at least eight different cell functions simultaneously. Bhaskaran et al. applied proteomic analysis (2–DE) to a human breast epithelial cell line after exposure to TGFb for various periods. Changes were detected by monitoring incorporation of 35[S–]methionine and 35[S–]cysteine compared to silver stain. In MCF10A cells, 1200 proteins were identified by staining and 1400 spots by metabolic labeling. Interestingly, not just one regulatory protein was activated per pathway, asmany as 13 regulators from both positive and negative complimentary functions were activated (e.g. pro– and anti–proliferative). Fromhere the story just gets better. Erk! Src!Bhaskaran,N. et al., Proteomics 2008, 8, 4507–4520.Protein microarrays and stopwatchesReality intrudes upon our research from time to time, like it or not, usually when someone from outside says “Really?” with a certain tone of voice that means “Can you prove that statement?” Korf et al. describe here a wellgrounded procedure for determining theamount and rate of phosphorylation of regulatory factors. The microarray format of the antibody capture probe assay minimizes the amount of protein required (about 5% of that needed for immunoblots) and the protocol assures reliable results through a careful matching of capture and reporter antibody pairs. The researchers estimate that less than 5 6 104 cells are required for phosphoprotein detection. Kinetics ofphosphorylationwere followedbysampling at intervalsof aminute. The systems analyzed were Erk1/2 and cell line BaF3, an erythropoetin receptor positive line, and fetal mouse liver erythroid colony forming cells. A software package based on R was customized for analysis of protocol data.Korf, U. et al., Proteomics 2008, 8, 4603–4612.

Cover Picture: Proteomics 21/2008

AbstractIn this issue of Proteomics you will find the following highlighted articles:When doing your best is too goodSometimes it pays to go back and question your assumptions, particularly if you are about to embark on a project with a lot of samples to process and analyze and, more particularly, if the instrumentation has changed substantially in the mean time. Villén et al. found a surprise at the end of their reevaluation of phosphopeptide identification. To start, they thought that the availability of 2–D and 3–D ion traps and high accuracy, high sensitivity would speed the process along substantially. Not so. First, neutral losses threatened to swamp out the MS2 spectrum from the 3–D ion trap, then they almost washed out the b– and y–type ions that comprise a critical internal ruler. The overabundance could be attenuated by using a new data dependent neutral loss MS3 method with 3–D ion traps. However, switching to high resolution ion traps reduced spectrum collection speed. . . Compromise time.Villén, J et al., Proteomics 2008, 8, 4444–4452.TGFbeta: a triskadecapoidal growth/ungrowth factor?One thing proteins do much more effectively than nucleic acids is multi–tasking. But they also start with a much larger tool chest – 20 amino acids vs. 4 nucleotides; dozens of post–translational modification possibilities vs. a few methylation sites. . . Transforming growth factor b takes advantage of its flexibility very effectively in regulating at least eight different cell functions simultaneously. Bhaskaran et al. applied proteomic analysis (2–DE) to a human breast epithelial cell line after exposure to TGFb for various periods. Changes were detected by monitoring incorporation of 35[S–]methionine and 35[S–]cysteine compared to silver stain. In MCF10A cells, 1200 proteins were identified by staining and 1400 spots by metabolic labeling. Interestingly, not just one regulatory protein was activated per pathway, asmany as 13 regulators from both positive and negative complimentary functions were activated (e.g. pro– and anti–proliferative). Fromhere the story just gets better. Erk! Src!Bhaskaran,N. et al., Proteomics 2008, 8, 4507–4520.Protein microarrays and stopwatchesReality intrudes upon our research from time to time, like it or not, usually when someone from outside says “Really?” with a certain tone of voice that means “Can you prove that statement?” Korf et al. describe here a wellgrounded procedure for determining theamount and rate of phosphorylation of regulatory factors. The microarray format of the antibody capture probe assay minimizes the amount of protein required (about 5% of that needed for immunoblots) and the protocol assures reliable results through a careful matching of capture and reporter antibody pairs. The researchers estimate that less than 5 6 104 cells are required for phosphoprotein detection. Kinetics ofphosphorylationwere followedbysampling at intervalsof aminute. The systems analyzed were Erk1/2 and cell line BaF3, an erythropoetin receptor positive line, and fetal mouse liver erythroid colony forming cells. A software package based on R was customized for analysis of protocol data.Korf, U. et al., Proteomics 2008, 8, 4603–4612.

Arsenic-induced aberrant gene expression in fetal mouse primary liver-cell cultures.

Exposure of maternal mice to inorganic arsenic through the drinking water induces liver tumors and aberrant gene expression in offspring when they reach adulthood. To help define if these are direct fetal effects of arsenic, fetal liver cells were isolated from untreated mice at gestation day 13.5 by mechanical dissection and centrifugation. Two hours after seeding the cells on collagen1-coated plates in William E media containing 10% fetal bovine serum, 1x ITS (insulin, transferrin, and selenium) and antibiotics, inorganic arsenite (0, 0.1, 0.3, and 1.0 microM) was added to the fresh media for 72 h. Cell morphology and viability were not significantly altered by these arsenic concentrations. At the end of arsenic exposure, cells were harvested into Trizol, and total RNA was extracted, purified, and subjected to real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Arsenite exposure produced a concentration-dependent induction of heme oxygenase-1 (up to eight-fold) and metallothionein-1 (up to five-fold), indicative of stress response to adapt to arsenic insult. Expression of genes related to steroid metabolism, such as 17beta-hydroxysteroid dehydrogenase-7 (HSD17beta7) and Cyp2a4, were increased approximately two-fold, together with increases in estrogen receptor-alpha (ER-alpha) and ER-alpha-linked genes, such as anterior gradient-2, keratin 1-19, and trefoil factor-3. Arsenic in vitro induced a three-fold increase in the expression of alpha-fetoprotein (AFP), a biomarker associated with transplacental arsenic-induced mouse liver tumors. Thus, exposure of mouse fetal liver cells to arsenic induces adaptive responses and aberrant gene expression, which could alter genetic programming at a very early life stage, potentially contributing to tumor formation much later in life.

Efficient Hematopoietic Differentiation of Human Embryonic Stem Cells on Stromal Cells Derived from Hematopoietic Niches

Hematopoietic stem cells derived from human embryonic stem cells (hESCs) could provide a therapeutic alternative to bone marrow transplants, but the efficiency of currently available derivation protocols is low. In this study, we investigated whether coculture with monolayers of cells derived from mouse AGM and fetal liver, or with stromal cell lines derived from these tissues, can enhance hESC hematopoietic differentiation. We found that under such conditions hESC-derived differentiating cells formed early hematopoietic progenitors, with a peak at day 18-21 of differentiation that corresponded to the highest CD34 expression. These hESC-derived hematopoietic cells were capable of primary and secondary hematopoietic engraftment into immunocompromised mice at substantially higher levels than described previously. Transcriptional and functional analysis identified TGF-beta1 and TGF-beta3 as positive enhancers of hESC hematopoietic differentiation that can further stimulate this process when added to the culture. Overall, our findings represent significant progress toward the goal of deriving functional hematopoietic stem cells from hESCs.