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Koichi Akashi

Publications and source records attributed to Koichi Akashi.

51 records · Page 3Linked to original sources
PubMed2003

GATA-1 converts lymphoid and myelomonocytic progenitors into the megakaryocyte/erythrocyte lineages.

GATA-1 is an essential transcription factor for megakaryocyte and erythrocyte (MegE) development. Here we show that hematopoietic progenitors can be reprogrammed by the instructive action of GATA-1. Enforced expression of GATA-1 in hematopoietic stem cells led to loss of self-renewal activity and the exclusive generation of MegE lineages. Strikingly, ectopic GATA-1 reprogrammed common lymphoid progenitors as well as granulocyte/monocyte (GM) progenitors to differentiate into MegE lineages, while inhibiting normal lymphoid or GM differentiation. GATA-1 upregulated critical MegE-related transcription factors such as FOG-1 and GATA-2 in lymphoid and GM progenitors, and their MegE development did not require "permissive" erythropoietin signals. Furthermore, GATA-1 induced apoptosis of proB and myelomonocytic cells, which could not be prevented by enforced permissive Bcl-2 or myeloid cytokine signals. Thus, GATA-1 specifically instructs MegE commitment while excluding other fate outcomes in stem and progenitor cells, suggesting that regulation of GATA-1 is critical in maintaining multilineage homeostasis.

Animals↗
PubMed2003

Hematopoietic stem cells expressing the myeloid lysozyme gene retain long-term, multilineage repopulation potential.

Single cell PCR studies showed that hematopoietic stem cells (HSCs) express a variety of lineage-affiliated genes. However, it remains unclear whether these cells exhibiting "lineage priming" represent bona fide stem cells or a subpopulation earmarked for differentiation. Here we have used a Cre-Lox approach to follow the fate of cells expressing a lineage-affiliated marker. We crossed lysozyme Cre mice with yellow fluorescent protein (EYFP) reporter mice and found EYFP gene expression not only in myelomonocytic cells but also in a fraction of HSCs as well as B cells and T cells. Transplantation of EYFP+ HSCs into primary and secondary recipients generated mice in which all hematopoietic cells were EYFP+. In contrast, crosses between CD19 Cre and lck Cre mice with reporter mice showed no EYFP expression in HSCs or intermediate progenitors. Our results demonstrate that lysozyme expression does not mark myeloid commitment and that long-term repopulation potential is maintained in primed HSCs.

Animals↗
PubMed2003

Unraveling the molecular components and genetic blueprints of stem cells.

Remarkable progress in stem cell biology research over the past few years has provoked a promise for the future of tissue regeneration and gene therapies; so much so, that the use of stem cells in clinical therapy seemed to be just around the corner. However, we now realize there is still a huge task before us to improve our understanding of the nature of stem cells before utilizing them to benefit human health. Stem cell behavior is determined by specific gene products; thus, unraveling the molecular components and genetic blueprints of stem cells will provide important insight into understanding stem cell properties. Here we summarize the research of various groups using microarray technology and other approaches to determine the gene expression profiles in stem cells, particularly in hematopoietic stem cells (HSCs). These works have, to a certain degree, helped to narrow down the candidate genes predominantly expressed in HSCs, revealed a list of stemness genes, and indirectly demonstrated the wide-open chromatin state of stem cells and, with it, the molecular basis of the multipotentiality of stem cells.

Animals↗
PubMed2002

Distinguishable live erythroid and myeloid cells in beta-globin ECFP x lysozyme EGFP mice.

We previously described a mouse line that contains green myelomonocytic cells due to the knock-in of enhanced green fluorescence protein (EGFP) into the lysozyme M gene.(1) We have now created a transgenic line with fluorescent erythroid cells using a beta-globin locus control region driving the enhanced cyan fluorescence protein (ECFP) gene. These mice exhibit cyan fluorescent cells specifically in the erythroid compartment and in megakaryocyte-erythroid progenitors. Crossing the animals with lysozyme EGFP mice yielded a line in which live erythroid and myeloid cells can readily be distinguished by fluorescence microscopy and by fluorescence-activated cell-sorter scanner. This cross allowed unambiguous identification of unstained mixed erythroid-myeloid colonies for the first time. The new mouse lines should become useful tools to dissect the branching between erythroid and myelomonocytic cells during in vitro differentiation of definitive multipotent progenitors.

Animals↗
PubMed2002

Transcriptional accessibility for genes of multiple tissues and hematopoietic lineages is hierarchically controlled during early hematopoiesis.

Hematopoietic stem cells (HSCs) maintain hematopoiesis by giving rise to all types of blood cells. Recent reports suggest that HSCs also possess the potential to generate nonhematopoietic tissues. To evaluate the underlying mechanisms in the commitment of HSCs into multitissue and multihematopoietic lineages, we performed oligonucleotide array analyses targeting for prospectively purified HSCs, multipotent progenitors (MPPs), common lymphoid progenitors (CLPs), and common myeloid progenitors (CMPs). Here we show that HSCs coexpress multiple nonhematopoietic genes as well as hematopoietic genes; MPPs coexpress myeloid and lymphoid genes; CMPs coexpress myeloerythroid, but not lymphoid genes, whereas CLPs coexpress T-, B-, and natural killer-lymphoid, but not myeloid, genes. Thus, the stepwise decrease in transcriptional accessibility for multilineage-affiliated genes may represent progressive restriction of developmental potentials in early hematopoiesis. These data support the hypothesis that stem cells possess a wide-open chromatin structure to maintain their multipotentiality, which is progressively quenched as they go down a particular pathway of differentiation.

Animals↗
PubMed2002

Prospective isolation of human clonogenic common myeloid progenitors.

The hierarchical development from hematopoietic stem cells to mature cells of the hematolymphoid system involves progressive loss of self-renewal capacity, proliferation ability, and lineage potentials. Here we show the prospective isolation of early developmental intermediates, the human clonogenic common myeloid progenitors and their downstream progeny, the granulocyte/macrophage and megakaryocyte/erythrocyte progenitors. All three populations reside in the lineage-negative (lin(-)) CD34(+)CD38(+) fraction of adult bone marrow as well as in cord blood. They are distinguishable by the expression of the IL-3R alpha chain, the receptor of an early-acting hematopoietic cytokine, and CD45RA, an isoform of a phosphotyrosine phosphatase involved in negative regulation of cytokine signaling. Multipotent progenitors, early lymphoid progenitors, and the here-defined myeloid progenitors express distinct profiles of hematopoiesis-affiliated genes. The isolation of highly purified hematopoietic intermediates provides tools to better understand developmental programs underlying normal and leukemic hematopoiesis.

Adult↗
PubMed2002

Distal elements are critical for human CD34 expression in vivo.

The elements regulating gene expression in hematopoietic stem cells are still poorly understood. We previously reported that a 141-kilobase (kb) human CD34 transgene confers properly regulated human CD34 expression in transgenic mice. A construct with only the human CD34 promoter and 3' enhancer region is not sufficient, suggesting that critical distal elements are necessary for expression of the human CD34 gene. To further localize such elements, we analyzed deletion constructs of the human CD34 gene and evaluated their function in transgenic mice. Constructs harboring as little as 18 kb of 5' and 26 kb of 3' human CD34 flanking sequence conferred human expression in tissues of transgenic mice with a pattern similar to that of the 141-kb human transgene. In contrast, a construct harboring 10 kb of 5' and 17 kb of 3' human CD34 flanking sequence gave no expression. These data demonstrate that regions between 10 to 18 kb upstream and/or 17 to 26 kb downstream of the human CD34 gene contain critical elements for human CD34 expression in vivo. Further functional analysis of these regions in transgenic mice will be crucial for understanding CD34 gene expression in hematopoietic stem and progenitor cells.

5' Flanking Region↗
PubMed2002

Transgenic targeting with regulatory elements of the human CD34 gene.

The human CD34 gene is expressed on early progenitor and stem cells in the bone marrow. Here we report the isolation of the human CD34 locus from a human P1 artificial chromosome (PAC) library and the characterization and evaluation of this genomic fragment for expression of reporter genes in stable cell lines and transgenic mice. We show that a 160-kb fragment spanning 110 kb of the 5' flanking region and 26 kb of the 3' flanking region of the CD34 gene directs expression of the human CD34 gene in the bone marrow of transgenic mice. The expression of human CD34 transgenic RNA in tissues was found to be similar to that of the endogenous murine CD34 gene. Colony-forming cell assays showed that bone marrow cells staining positive for human CD34 consist of early progenitor cells in which expression of CD34 decreased with cell maturation. In order to test the construct for its ability to express heterologous genes in vivo, we used homologous recombination in bacteria to insert the tetracycline-responsive transactivator protein tTA. Analysis of transgenic human CD34-tTA mice by cross breeding with a strain carrying Cre recombinase under control of a tetracycline-responsive element demonstrated induction of Cre expression in mice in a pattern consistent with the expression of the human CD34 transgene.

3' Flanking Region↗
PubMed2002

Myeloid progenitors protect against invasive aspergillosis and Pseudomonas aeruginosa infection following hematopoietic stem cell transplantation.

Myelotoxic treatments for oncologic diseases are often complicated by neutropenia, which renders patients susceptible to potentially lethal infections. In these studies of murine hematopoietic stem cell transplantation (HSCT), cotransplantation of lineage-restricted progenitors known as common myeloid progenitors (CMP) and granulocyte-monocyte progenitors (GMP) protects against death following otherwise lethal challenge with either of 2 pathogens associated with neutropenia: Aspergillus fumigatus and Pseudomonas aeruginosa. Cotransplantation of CMP/GMP resulted in a significant and rapid increase in the absolute number of myeloid cells in the spleen, most of which were derived from the donor CMP/GMP. Despite persistent peripheral neutropenia, improved survival correlated with the measurable appearance of progenitor-derived myeloid cells in the spleen. A marked reduction or elimination of tissue pathogen load was confirmed by culture and correlated with survival. Localization of infection by P aeruginosa and extent of disease was also assessed by in vivo bioluminescent imaging using a strain of P aeruginosa engineered to constitutively express a bacterial luciferase. Imaging confirmed that transplantation with a graft containing hematopoietic stem cells and CMP/GMP reduced the bacterial load as early as 18 hours after infection. These results demonstrate that enhanced reconstitution of a tissue myeloid pool offers protection against lethal challenge with serious fungal and bacterial pathogens.

Administration, Inhalation↗
PubMed2002

Induction of granulocytic differentiation by 2 pathways.

The CCAAT enhancer binding protein alpha (C/EBP alpha) transcription factor plays a critical role in granulocytopoiesis. Mice with a disruption of the C/EBP alpha gene demonstrate an early block in granulocytic differentiation, and disruption of C/EBP alpha function is a common theme in many types of human acute myelogenous leukemia, which is characterized by a block in myeloid development. To characterize further the nature of this block, we derived cell lines from the fetal liver of C/EBP alpha-deficient animals. These lines resembled morphologically the immature myeloid blasts observed in C/EBP alpha(-/-) fetal livers and did not express messenger RNA encoding early myeloid genes such as myeloperoxidase. Similarly, granulocytic markers such as Mac-1 and Gr-1 were not expressed; nor were erythroid and lymphoid surface antigens. Introduction of an inducible C/EBP alpha gene into the line revealed that conditional expression of C/EBP alpha induced the C/EBP family members C/EBP beta and C/EBP epsilon and subsequent granulocyte differentiation. Similar results were obtained when C/EBP alpha(-/-) cells were stimulated with the cytokines interleukin-3 and granulocyte-macrophage colony-stimulating factor, but not with all-trans retinoic acid, supporting a model of at least 2 pathways leading to the differentiation of myeloid progenitors to granulocytes and implicating induction of other C/EBP family members in granulopoiesis.

Animals↗
PubMed2002

Differential regulation of the human and murine CD34 genes in hematopoietic stem cells.

Human CD34 (hCD34)-positive cells are used currently as a source for hematopoietic transplantation in humans. However, in steady-state murine hematopoiesis, hematopoietic stem cells (HSCs) with long-term reconstitution activity are found almost exclusively in the murine CD34 (mCD34)-negative to low fraction. To evaluate the possible differences in hCD34 and mCD34 gene expression in hematopoiesis, we made transgenic mouse strains with human genomic P1 artificial chromosome clones spanning the entire hCD34 genomic locus. In all transgenic mouse strains, a vast majority of phenotypic and functional HSC populations including mCD34(-/lo) express the hCD34 transgene. These data strongly support the notion that hCD34(+) human bone marrow cells contain long-term HSCs that can maintain hematopoiesis throughout life.

Animals↗
PubMed2002

Myeloid or lymphoid promiscuity as a critical step in hematopoietic lineage commitment.

We demonstrate here that "promiscuous" expression of myeloid or lymphoid genes precedes lineage commitment in hematopoiesis. Prospectively purified single common myeloid progenitors (CMPs) coexpress myelo-erythroid but not lymphoid genes, whereas single common lymphoid progenitors (CLPs) coexpress T and B lymphoid but not myeloid genes. Genes unrelated to the adopted lineage are downregulated in bipotent and monopotent descendants of CMPs and CLPs. Promiscuous gene expression does not alter the biological potential of multipotent progenitors: CMPs with an activated endogenous M lysozyme locus yield normal proportions of myelo-erythroid colonies, and CLPs expressing the pre-T cell receptor alpha gene differentiate into normal numbers of B cells. Thus, the accessibility for multiple myeloid or lymphoid programs promiscuously may allow flexibility in fate commitments at these multipotent stages.

Animals↗
PubMed2002

Slug, a highly conserved zinc finger transcriptional repressor, protects hematopoietic progenitor cells from radiation-induced apoptosis in vivo.

We show here that a zinc finger transcriptional repressor, Slug, which is aberrantly upregulated by the E2A-HLF oncoprotein in pro-B cell acute leukemia, functions as an antiapoptotic factor in normal hematopoietic progenitor cells. Slug(-/-) mice were much more radiosensitive than wild-type mice, dying earlier and showing accentuated decreases in peripheral blood cell counts, as well as abundant microhemorrhages and widely disseminated bacterial microabscesses throughout the body. Slug expression was detected in diverse subsets of hematopoietic progenitors, but not in more differentiated B and T lymphoid cells, and there was a significant increase in apoptotic (TUNEL-positive) bone marrow progenitor cells in irradiated Slug(-/-) mice compared to wild-type controls. These results implicate Slug in a novel survival pathway that protects hematopoietic progenitors from apoptosis after DNA damage.

Animals↗
PubMed2002

Myeloerythroid-restricted progenitors are sufficient to confer radioprotection and provide the majority of day 8 CFU-S.

Whole-body irradiation at the minimal lethal dose causes bone marrow failure and death within 12-18 days. To identify the principal components of the hematopoietic system that are radioprotective, we transplanted lethally irradiated mice with purified progenitors: common myeloid progenitors (CMPs), megakaryocyte/erythrocyte-restricted progenitors (MEPs), or granulocyte/monocyte-restricted progenitors (GMPs). Transplanted CMPs gave rise to cells both of the granulocyte/monocyte (GM) series and the megakaryocyte/erythrocyte series, whereas GMPs or MEPs showed reconstitution of only GM or ME cells, respectively. CMPs and MEPs but not GMPs protected mice in a dose-dependent manner, suggesting that erythrocytes, platelets, or both are the critical effectors of radioprotection. Accordingly, CMPs and MEPs formed robust colonies in recipient bone marrow and spleen, whereas GMPs formed small colonies that rapidly disappeared. Direct comparisons of spleen CFU (CFU-S) potentials among each progenitor subset showed that MEPs contain the vast majority of day 8 CFU-S activity, suggesting that day 8 CFU-S are the precursors of radioprotective cell subsets. All animals radioprotected for 30 days subsequently survived for at least 6 months post-transplant, and showed only host-derived hematopoiesis after 30 days. These findings suggest that rare hematopoietic stem cells survive myeloablation that can eventually repopulate irradiated hosts if myeloerythroid-restricted progenitors transiently rescue ablated animals through the critical window of bone marrow failure.

Animals↗