Metabolic Programming of the Fetal Liver Hematopoietic Stem and Progenitor Cell Pool

Abstract Evidence in several organ systems demonstrates that pregnancy presents a window of vulnerability for establishing a foundation for health or chronic disease. Overnutrition and the complex metabolic changes that can accompany it can result in permanent phenotypic changes and a predisposition to metabolic syndrome, inflammatory or immune-mediated diseases. We previously reported that prenatal overnutrition stunted fetal liver size. Herein, we hypothesize that this might perturb hematopoietic stem and progenitor cell (HSPC) expansion. To test the effects of a high-fat diet (HFD) and maternal obesity on offspring hematopoiesis, we used a mouse model of diet-induced obesity, feeding female mice a HFD or control diet starting at 5-7 weeks of age and keeping them on the respective diet during subsequent breeding and pregnancy. We then studied offspring at gestational day 14.5 by immunophenotyping, gene expression analysis, qRT-PCR, and transplantation. Fetal livers from HFD offspring had 51% fewer c-Kit+ Sca-1+ Linlo/- and 27% fewer AA4.1+ Sca-1+ Linlo/- (ASL) hematopoietic stem and progenitor cells (HSPC) than controls. This restriction in HSPC numbers was not due to apoptosis or increased reactive oxygen species, as tested by flow cytometry. To determine whether there might be an increase in hematopoietic differentiation to account for relative HSPC deficiencies in HFD livers, we examined hematopoietic lineage subsets. HFD fetal livers had a relative increase in myeloid (Gr-1+/Ter119+) and B220+ lymphoid cells, with comparable proportion of CD3+ cells to controls. Taken together, these results suggest that chronic HFD fetal programming skews fetal liver HSPCs toward differentiation. When we examined global gene expression of male HFD fetal livers versus controls by RNA-seq, we found differential expression of 125 genes. Among the upregulated transcripts, several were involved in hematopoietic regulation, stress response, and HSPC migration. We then used qRT-PCR to test for expression of several of these genes, along with genes critically involved in fetal HSPC self-renewal, within an HSPC-enriched (Sca-1+) population of chronic HFD fetal liver cells. As in RNA-seq, Matrixmetalloproteinase-8 and 9 (Mmp8, Mmp9), which are involved in cell mobilization, were upregulated in HFD-programmed cells. Early growth response-1 (Egr-1) was downregulated as well, further suggesting premature migration of HSPCs from HFD fetal liver. Hmga2, which is implicated in fetal stem cell self-renewal, and its direct target, Igf2bp2, were significantly downregulated in chronic HFD Sca-1+ cells. Along with the immunophenotyping data, these findings suggest that maternal obesity and HFD bias HSPCs toward differentiation, at the expense of self-renewal. To dissect the direct metabolic impact, we studied fetal livers from timed pregnancy cohorts after acute HFD exposure or diet reversal in obese dams, which partially ameliorated several molecular and immunophenotypic endpoints. Finally, we performed a functional test of chronic HFD fetal liver cells by transplantation. A non-competitive transplant into irradiated male recipients yielded no difference in chimerism between HFD or control fetal liver-engrafted animals. Next, we preconditioned a cohort of female and male animals on HFD (or control diet) for 11 weeks, irradiated them, and then competitively transplanted them with a 1:1 ratio of HFD and control fetal liver cells. HFD-programmed fetal liver HSPCs engrafted HFD-conditioned male recipients at significantly lower rates than in HFD-conditioned female or control recipients of either sex. HFD-programmed donor cells retained the significant bias toward the myeloid (Gr-1+/Mac-1+) lineage, noted in the primary graft cells, and away from the B220+ B cell lineage in HFD-conditioned males. In aggregate, prenatal HFD and maternal obesity suppress self-renewal in favor of HSPC differentiation during a time of critical developmental expansion. This suggests an HSPC defect that appears at least partly specified by the stem cell microenvironment. Our work is the first to demonstrate metabolic vulnerability of the hematopoietic stem and progenitor cell compartment and establishes the hematopoietic system as a target for in utero developmental programming. Disclosures No relevant conflicts of interest to declare.

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Bclaf1 Promotes Maintenance and Self-Renewal of Fetal Hematopoietic Stem Cells

Blood ◽

10.1182/blood-2018-99-114144 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1269-1269 ◽

Cited By ~ 1

Author(s):

Lynn S. White ◽

Deepti Soodgupta ◽

Rachel L. Johnston ◽

Jeffrey A. Magee ◽

Jeffrey J. Bednarski

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Liver Cells ◽

Lower Percentage ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

Fetal Liver Cells

Abstract Hematopoietic stem cells (HSC) persist throughout life by undergoing extensive self-renewal divisions while maintaining an undifferentiated state. The mechanisms that support HSC self-renewal change throughout the course of development as temporal changes in transcriptional regulators coordinate distinct genetic programs in fetal, post-natal and adult HSCs. These self-renewal programs are often ectopically activated in leukemia cells to drive neoplastic proliferation and high expression of HSC-associated genes predicts a poor prognosis in acute myelogenous leukemia (AML). In this regard, it was recently shown that expression of the transcriptional regulator BCLAF1 (Bcl2 associated transcription factor 1) is increased in AML blasts relative to normal precursor populations and suppression of BCLAF1 causes reduced proliferation and induction of differentiation to a dendritic cell fate. These findings raise the question of whether BCLAF1 may regulate normal as well as neoplastic self-renewal programs. We find that Bclaf1 is highly expressed in HSCs versus committed bone marrow populations consistent with a potential role for this gene in HSC functions. To test whether BCLAF1 regulates HSC development and hematopoiesis, we used germline loss of function mice. Bclaf1-/- mice succumb to pulmonary failure shortly after birth due to poor lung development, so we assessed prenatal hematopoiesis. Bclaf1-deficient mice had significantly reduced HSC and hematopoietic progenitor cell (HPC) frequencies and numbers despite normal fetal liver cellularity. To determine if Bclaf1 is required for HSC function during fetal development, we performed competitive reconstitution assays. Fetal liver cells from Bclaf1+/+or Bclaf1-/-mice were transplanted along with wild-type competitor bone marrow cells into lethally irradiated recipient mice. Compared to recipients of Bclaf1+/+fetal liver cells, recipients of Bclaf1-/-cells had a significantly lower percentage of donor-derived leukocytes at all time points after transplantation as well as reduced percentage of donor HSCs at 16 weeks post-transplant. Notably, all leukocyte populations (B cells, T cells, granulocytes and macrophages) from Bclaf1-/-donors were reduced consistent with an abnormality in HSC repopulating activity rather than a defect in a specific differentiation pathway. Consistent with these findings, Bclaf-deficient cells did not engraft in competitive transplants with limiting numbers of sorted fetal liver HSCs whereas sorted wild-type Bclaf1+/+cells effectively reconstituted hematopoiesis in recipient mice. In addition, Vav-cre:Bclaf1flox/floxmice, which have selective deletion of Bclaf1 in hematopoietic cells, have reduced frequencies and numbers of fetal liver HSCs identical to the findings observed in germline Bclaf1-/-mice. These results show that loss of Bclaf1 leads to defective development and repopulating activity of fetal HSCs. Interestingly, when adult mice are successfully engrafted with Bclaf1-deficient HSCs, the donor HSCs suffer no additional functional impairment. Furthermore, in secondary transplant experiments Bclaf1-deficient HSCs maintain long-term repopulating activity. Thus, Bclaf1 may have distinct functions in fetal versus adult HSC self-renewal. Collectively, our findings reveal Bclaf1 is a novel regulator of fetal HSC function and suggest that it may have distinct functions in different developmental contexts. Disclosures No relevant conflicts of interest to declare.

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Roles of Histone Acetyltransferase MORF and MOZ in Hematopoiesis.

Blood ◽

10.1182/blood.v114.22.2525.2525 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2525-2525

Author(s):

Takuo Katsumoto ◽

Issay Kitabayashi

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Fetal Liver ◽

Liver Cells ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

Fetal Liver Cells

Abstract Abstract 2525 Poster Board II-502 MOZ (MOnocytic leukemia Zinc finger protein) and MORF (MOz Related Factor), Myst-type histone acetyltransferases, are involved in chromosome translocations associated with FAB-M4/5 subtypes of acute myeloid leukemia. We have reported that MOZ is essential for hematopoietic cell development and self-renewal of hematopoietic stem cells. To explore the possibility MORF also plays important roles in hematopoiesis, we generated Morf-deficient mice with homologous recombination methods. Morf−/− mice were smaller than their wildtype littermates and died within 4 weeks after birth on C57BL/6 background. In MORF−/− fetal liver, Flt3-negative KSL (c-Kit+ Sca-1+ Lineage-) cells containing hematopoietic stem cells were decreased. When fetal liver cells were transplanted into irradiated recipient mice, MORF−/− cells less efficiently reconstituted hematopoiesis than wild-type cells. Additionally, bone marrow cells reconstituted with MORF−/− cells rarely contributed to hematopoiesis in secondary transplants. To reveal relationship between MORF and MOZ in hematopoiesis, we generated double heterozygous (Moz+/− Morf+/−) mouse. Double heterozygous mice were smaller than wild-type littermates and died at least 4 weeks after birth. Numbers of KSL cells, especially Flt3- KSL cells and common myeloid progenitors were decreased in the double heterozygous embryos. The double heterozygous fetal liver cells also displayed less activity to reconstitute hematopoiesis than MOZ+/− or MORF+/− cells. Since MORF−/− mice and MOZ/MORF double heterozygous mice were alive at adult on a mixed C57BL/6/DBA2 genetic background, we investigated adult hematopoiesis in these mice. MORF−/− or MOZ/MORF double heterozygous mice were smaller than their wild-type littermates and had small numbers of thymocytes and splenocytes. However, there were no significant differences in number of bone marrow cells and hematopoietic lineage population in MORF−/− or MOZ/MORF double heterozygous mice. These results suggest that MORF as well as MOZ plays important roles in self-renewal of hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

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Hematopoietic Defects in Cited2 Mutant Fetal Liver.

Blood ◽

10.1182/blood.v106.11.2272.2272 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2272-2272

Author(s):

Yu Chen ◽

Yu-Chung Yang

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Liver Cells ◽

Hematopoietic Progenitors ◽

Self Renewal ◽

Hematopoietic Stem ◽

Fetal Liver Cells

Abstract 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.

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Elevated Mcl-1 perturbs lymphopoiesis, promotes transformation of hematopoietic stem/progenitor cells, and enhances drug resistance

Blood ◽

10.1182/blood-2010-04-281071 ◽

2010 ◽

Vol 116 (17) ◽

pp. 3197-3207 ◽

Cited By ~ 80

Author(s):

Kirsteen J. Campbell ◽

Mary L. Bath ◽

Marian L. Turner ◽

Cassandra J. Vandenberg ◽

Philippe Bouillet ◽

...

Keyword(s):

Progenitor Cells ◽

Progenitor Cell ◽

Fetal Liver ◽

Cytotoxic Agents ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Fetal Liver Cells ◽

Follicular Lymphomas ◽

The Impact

Abstract Diverse human cancers with poor prognosis, including many lymphoid and myeloid malignancies, exhibit high levels of Mcl-1. To explore the impact of Mcl-1 overexpression on the hematopoietic compartment, we have generated vavP-Mcl-1 transgenic mice. Their lymphoid and myeloid cells displayed increased resistance to a variety of cytotoxic agents. Myelopoiesis was relatively normal, but lymphopoiesis was clearly perturbed, with excess mature B and T cells accumulating. Rather than the follicular lymphomas typical of vavP-BCL-2 mice, aging vavP-Mcl-1 mice were primarily susceptible to lymphomas having the phenotype of a stem/progenitor cell (11 of 30 tumors) or pre-B cell (12 of 30 tumors). Mcl-1 overexpression dramatically accelerated Myc-driven lymphomagenesis. Most vavP-Mcl-1/ Eμ-Myc mice died around birth, and transplantation of blood from bitransgenic E18 embryos into unirradiated mice resulted in stem/progenitor cell tumors. Furthermore, lethally irradiated mice transplanted with E13 fetal liver cells from Mcl-1/Myc bitransgenic mice uniformly died of stem/progenitor cell tumors. When treated in vivo with cyclophosphamide, tumors coexpressing Mcl-1 and Myc transgenes were significantly more resistant than conventional Eμ-Myc lymphomas. Collectively, these results demonstrate that Mcl-1 overexpression renders hematopoietic cells refractory to many cytotoxic insults, perturbs lymphopoiesis and promotes malignant transformation of hematopoietic stem and progenitor cells.

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Expansion of mouse hematopoietic stem/progenitor cells in three-dimensional cocultures on growth-suppressed stromal cell layer

The International Journal of Artificial Organs ◽

10.1177/0391398819827596 ◽

2019 ◽

Vol 42 (7) ◽

pp. 374-379 ◽

Cited By ~ 1

Author(s):

Hirotoshi Miyoshi ◽

Chiaki Sato ◽

Yuichiro Shimizu ◽

Misa Morita

Keyword(s):

Progenitor Cells ◽

Mitomycin C ◽

Stromal Cells ◽

Fetal Liver ◽

Three Dimensional ◽

Hematopoietic Cells ◽

Liver Cells ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Fetal Liver Cells

With the aim of establishing an effective method to expand hematopoietic stem/progenitor cells for application in hematopoietic stem cell transplantation, we performed ex vivo expansion of hematopoietic stem/progenitor cells derived from mouse fetal liver cells in three-dimensional cocultures with stromal cells. In these cocultures, stromal cells were first cultured within three-dimensional scaffolds to form stromal layers and then fetal liver cells containing hematopoietic cells were seeded on these scaffolds to expand the hematopoietic cells over the 2 weeks of coculture in a serum-containing medium without the addition of cytokines. Prior to coculture, stromal cell growth was suppressed by treatment with the DNA synthesis inhibitor mitomycin C, and its effect on hematopoietic stem/progenitor cell expansion was compared with that in control cocultures in which fetal liver cells were cocultured with three-dimensional freeze-thawed stromal cells. After coculture with mitomycin C-treated stromal cells, we achieved a several-fold expansion of the primitive hematopoietic cells (c-kit+hematopoietic progenitor cells >7.8-fold, and CD34+hematopoietic stem/progenitor cells >3.5-fold). Compared with control cocultures, expansion of hematopoietic stem/progenitor cells tended to be lower, although that of hematopoietic progenitor cells was comparable. Thus, our results suggest that three-dimensional freeze-thawed stromal cells have higher potential to expand hematopoietic stem/progenitor cells compared with mitomycin C-treated stromal cells.

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Expansion of hematopoietic stem cells in the developing liver of a mouse embryo

Blood ◽

10.1182/blood.v95.7.2284 ◽

2000 ◽

Vol 95 (7) ◽

pp. 2284-2288 ◽

Cited By ~ 210

Author(s):

Hideo Ema ◽

Hiromitsu Nakauchi

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Mouse Embryo ◽

Bone Marrow Cells ◽

Fetal Liver ◽

Cell Number ◽

Liver Cells ◽

Single Wave ◽

Hematopoietic Stem ◽

Fetal Liver Cells

Abstract The activity of hematopoietic stem cells in the developing liver of a C57BL/6 mouse embryo was quantified by a competitive repopulation assay. Different doses of fetal liver cells at days 11 to 18 of gestation were transplanted into irradiated mice together with 2 × 105 adult bone marrow cells. A long-term repopulation in myeloid-, B-cell, and T-cell lineage by fetal liver cells was evaluated at 20 weeks after transplantation. At day 12 of gestation multilineage repopulating activity was first detected in the liver as 50 repopulating units (RU) per liver. The number of RU per liver increased 10-fold and 33-fold by day 14 and day 16 of gestation, and decreased thereafter, suggesting a single wave of stem cell development in the fetal liver. A limiting dilution analysis revealed that the frequency of competitive repopulating units (CRU) in fetal liver cells at day 12 of gestation was similar to that at day 16 of gestation. Because of an increase of total fetal liver cell number, the absolute number of CRU per liver from days 12 to 16 of gestation increased 38-fold. Hence, the mean activity of stem cells (MAS) that is given by RU per CRU remained constant from days 12 to 16 of gestation. From these data we conclude that hematopoietic stem cells expand in the fetal liver maintaining their level of repopulating potential.

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Phenotypic and Functional Evidence for the Expression of CD4 by Hematopoietic Stem Cells Isolated From Human Fetal Liver

Blood ◽

10.1182/blood.v89.4.1364 ◽

1997 ◽

Vol 89 (4) ◽

pp. 1364-1375 ◽

Cited By ~ 48

Author(s):

Marcus O. Muench ◽

Maria Grazia Roncarolo ◽

Reiko Namikawa

Keyword(s):

Stem Cells ◽

Fetal Liver ◽

Liver Cells ◽

Proliferative Potential ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Human Fetal Liver ◽

Fetal Bone ◽

Fetal Stem Cells ◽

Fetal Liver Cells

Abstract Expression of the CD4 antigen was observed on human fetal liver, fetal bone marrow (BM), and umbilical cord blood progenitors expressing high levels of CD34. Using clonal and liquid-culture assays, CD4+ CD34++ Lin− (lineage = CD3, CD8, CD10, CD14, CD15, CD16, CD19, CD20, and glycophorin A) fetal liver progenitors were found to have a greater proliferative potential than CD4− CD34++ Lin− progenitors, whereas the CD4− fraction was more enriched for erythroid progenitors. Both the CD4+ and the CD4− progenitor subpopulations also gave rise to multilineage engraftment upon transplantation into human fetal bone fragments, supportive of B-lymphoid and myeloid growth, or into human fetal thymic fragments, supportive of T-cell growth, implanted in scid/scid (SCID) mice. However, in SCID-hu mice transplanted with graded doses of donor cells ranging from 2.0 × 102 to 2.0 × 104 cells, BM reconstitution by the CD4+ fraction of CD34++ Lin− cells was more frequent than by the CD4− fraction when low numbers of cells were injected. These functional data strongly suggest that stem cells reside among CD4+ CD34++ Lin− fetal liver cells. This hypothesis was further supported by the observations that CD4+ CD34++ Lin− fetal liver cells were enriched for CDw90+ (Thy-1), CD117+ (kit), CD123+, HLA-DR+, CD7−, CD38−, CD45RA−, CD71−, CD115− (fms), and rhodamine 123dull cells, a phenotypic profile believed to represent fetal stem cells. Furthermore, all CD4+ CD34++ Lin− fetal liver cells also expressed CD13 and CD33.

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Reduction of Core Binding Factor beta (CBFβ) Dosage Blocks T Cell Development.

Blood ◽

10.1182/blood.v106.11.2714.2714 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2714-2714

Author(s):

Yalin Guo ◽

Laleh Talebian ◽

Ivan Maillard ◽

Caroline L. Speck ◽

Warren S. Pear ◽

...

Keyword(s):

T Cell ◽

Dna Binding ◽

Fetal Liver ◽

T Cell Development ◽

Notch Pathway ◽

Cell Development ◽

Liver Cells ◽

Normal Numbers ◽

Hematopoietic Stem ◽

Fetal Liver Cells

Abstract Core binding factors (CBFs) are heterodimers consisting of a DNA binding subunit (Runx1, Runx2, or Runx3) and a non-DNA binding CBFβ subunit. CBFβ increases the affinity of the Runx subunits for DNA. Embryos deficient for Runx1 or CBFβ die at midgestation with a complete failure of definitive hematopoiesis due to a block in hematopoietic stem cell (HSC) emergence. To examine the role of core binding factors at later stages of hematopoiesis, we generated a hypomorphic Cbfb allele (Cbfbrss), that when carried over a Cbfb null allele (Cbfbrss/−) results in a 3-4 fold reduction in CBFβ protein levels. Although HSCs emerge in Cbfbrss/− animals, fetal T cell development is severely impaired. Here we examined the T cell developmental block in more detail by culturing fetal liver cells from Cbfbrss/− animals on OP9 stromal cells that express the Notch ligand Delta-like-1 (DL1) (Schmitt and Zúñiga-Pflücker, Immunity17: 749, 2002). Fetal livers (E14.5) from Cbfbrss/− animals contained normal numbers of both c-kit+Sca-1+lin- cells and c-kit+IL7r+ lymphoid progenitors. Lin- fetal liver progenitors cultured on OP9-DL1 cells in the presence of IL-7 and Flt3L displayed a growth disadvantage relative to wild type cells, and a block at the double negative 1 (DN1, CD44+ CD25−) stage of T cell development. The T cell defect could be rescued by retroviral transduction of the CBFβ heterodimerization domain into lin- fetal liver cells, but not by a G61A/N104A mutant that cannot bind the Runx subunits. Genes whose expression was decreased in DN1 cells purified from the OP9-DL1 cultures included CD3 and the early T cell transcription factors GATA3 and TCF. Although expression of several Notch pathway genes (Notch1, Hes-1/5, Deltex-1) was mildly decreased, Notch signals were clearly transduced, suggesting that Notch signaling was intact. These data demonstrate that reduced CBFβ levels impair the differentiation of stem cells/progenitors into T cells at the earliest stage of T cell development. This in vitro model will be useful for characterizing the molecular circuitry involving CBFβ in T cell development, and for identifying CBFβ protein partners.

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Roles of MOZ in Hematopoiesis.

Blood ◽

10.1182/blood.v106.11.2269.2269 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2269-2269

Author(s):

Takuo Katsumoto ◽

Yukiko Aikawa ◽

Takahiro Ochiya ◽

Issay Kitabayashi

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Fetal Liver ◽

Liver Cells ◽

Recipient Mouse ◽

Wild Type ◽

Hematopoietic Stem ◽

Homozygous Mutant ◽

Fetal Liver Cells ◽

Acute Myeloid

Abstract The AML1-CBFβ transcription factor complex is the most frequent target of specific chromosome translocations in acute myeloid leukemia (AML). The monocytic leukemia zinc finger (MOZ) gene, which encodes a MYST-type histone acetyltransferase (HAT), is also involved in leukemia-associated translocations such as t(8;16), t(8;22) and inv(8), which are associated with acute myeloid leukemia with M4/5 subtypes. We previously found that MOZ functions as a potent coactivator for AML1. To investigate roles of MOZ in normal hematopoiesis, we generated MOZ-deficient mice using gene-targeting method. MOZ homozygous mutant is embryonic lethal and it died between days 14 and 15 of gestation. In fetal liver of MOZ-deficient E14.5 embryos, the total cell numbers and the colony-forming cells (CFCs) in a methylcellulose medium were remarkably reduced when compared with wild-type littermates. Flow cytometry analysis indicated that hematopoietic stem cells (HSCs) and progenitors of both myeloid and lymphoid lineages were severely reduced in MOZ-deficient embryos. Especially, the levels of c-kit expression were strongly reduced in lineage-negative cells. Differentiation arrest of erythroid progenitors at a terminal stage and increase in the numbers of Mac-1 and Gr-1 positive cells suggest that MOZ also plays roles in cell differentiation of erythroid, monocytic and granulocytic lineages. In E12.5 MOZ deficient fetal liver cells, expression profile analysis revealed decreases in expressions of thrombopoietin receptor c-mpl, Wnt related ligand dkk2 and HoxA9 and increase in HoxA5 expression. To further determine roles of MOZ in HSCs functions and their progenitors differentiation ability, competitive reconstitution assays were performed. Ly5.2+ fetal liver cells from wild-type, heterozygous or homozygous mutant embryos together with Ly5.1+ competitor fetal liver cells were transplanted into γ-irradiated Ly5.1+/Ly5.2+ recipient mouse. Ly5.2+ wild-type cells were observed in recipient mice after transplantation. However, cells derived from MOZ homozygous mutant embryos were not detected in peripheral blood, bone marrow, spleen and thymus. Reduced population of cells derived from heterozygous mutant embryos were observed. These data suggest that MOZ is required for lymphoid and myeloid development and for self-renewal of HSCs.

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

Blood ◽

10.1182/blood-2005-10-4135 ◽

2006 ◽

Vol 108 (2) ◽

pp. 737-744 ◽

Cited By ~ 131

Author(s):

Injune Kim ◽

Shenghui He ◽

Ömer H. Yilmaz ◽

Mark J. Kiel ◽

Sean J. Morrison

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Liver Cells ◽

Stem Cell Markers ◽

Hematopoietic Stem ◽

Similar Expression Pattern ◽

Slam Family ◽

Fetal Liver Cells

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 ThylowSca-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.

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