Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 115
Filter
1.
EMBO J ; 39(12): e102930, 2020 06 17.
Article in English | MEDLINE | ID: mdl-32347571

ABSTRACT

During angiogenesis, VEGF acts as an attractive cue for endothelial cells (ECs), while Sema3E mediates repulsive cues. Here, we show that the small GTPase RhoJ integrates these opposing signals in directional EC migration. In the GTP-bound state, RhoJ interacts with the cytoplasmic domain of PlexinD1. Upon Sema3E stimulation, RhoJ released from PlexinD1 induces cell contraction. PlexinD1-bound RhoJ further facilitates Sema3E-induced PlexinD1-VEGFR2 association, VEGFR2 transphosphorylation at Y1214, and p38 MAPK activation, leading to reverse EC migration. Upon VEGF stimulation, RhoJ is required for the formation of the holoreceptor complex comprising VEGFR2, PlexinD1, and neuropilin-1, thereby preventing degradation of internalized VEGFR2, prolonging downstream signal transductions via PLCγ, Erk, and Akt, and promoting forward EC migration. After conversion to the GDP-bound state, RhoJ shifts from PlexinD1 to VEGFR2, which then terminates the VEGFR2 signals. RhoJ deficiency in ECs efficiently suppressed aberrant angiogenesis in ischemic retina. These findings suggest that distinct Rho GTPases may act as context-dependent integrators of chemotactic cues in directional cell migration and may serve as candidate therapeutic targets to manipulate cell motility in disease or tissue regeneration.


Subject(s)
Cell Movement , Endothelial Cells/metabolism , Signal Transduction , rho GTP-Binding Proteins/metabolism , Animals , Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Transgenic , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism , rho GTP-Binding Proteins/genetics
2.
Nat Rev Mol Cell Biol ; 9(9): 725-9, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18698329

ABSTRACT

Induced pluripotent stem (iPS) cells are human somatic cells that have been reprogrammed to a pluripotent state. There are several hurdles to be overcome before iPS cells can be considered as a potential patient-specific cell therapy, and it will be crucial to characterize the developmental potential of human iPS cell lines. As a research tool, iPS-cell technology provides opportunities to study normal development and to understand reprogramming. iPS cells can have an immediate impact as models for human diseases, including cancer


Subject(s)
Biomedical Research , Cell- and Tissue-Based Therapy , Pluripotent Stem Cells/cytology , Humans
3.
Nature ; 470(7335): 503-9, 2011 Feb 24.
Article in English | MEDLINE | ID: mdl-21326203

ABSTRACT

The neural fate is generally considered to be the intrinsic direction of embryonic stem (ES) cell differentiation. However, little is known about the intracellular mechanism that leads undifferentiated cells to adopt the neural fate in the absence of extrinsic inductive signals. Here we show that the zinc-finger nuclear protein Zfp521 is essential and sufficient for driving the intrinsic neural differentiation of mouse ES cells. In the absence of the neural differentiation inhibitor BMP4, strong Zfp521 expression is intrinsically induced in differentiating ES cells. Forced expression of Zfp521 enables the neural conversion of ES cells even in the presence of BMP4. Conversely, in differentiation culture, Zfp521-depleted ES cells do not undergo neural conversion but tend to halt at the epiblast state. Zfp521 directly activates early neural genes by working with the co-activator p300. Thus, the transition of ES cell differentiation from the epiblast state into neuroectodermal progenitors specifically depends on the cell-intrinsic expression and activator function of Zfp521.


Subject(s)
Cell Differentiation , Embryonic Stem Cells/cytology , Neural Stem Cells/cytology , Transcription Factors/metabolism , Animals , Bone Morphogenetic Protein 4/deficiency , Bone Morphogenetic Protein 4/genetics , Bone Morphogenetic Protein 4/metabolism , Cadherins/metabolism , Cell Lineage , Cells, Cultured , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Embryonic Stem Cells/metabolism , Gene Expression Regulation, Developmental/genetics , Germ Layers/cytology , Germ Layers/embryology , Germ Layers/metabolism , HEK293 Cells , Humans , Mice , Models, Biological , Neural Plate/cytology , Neural Plate/embryology , Neural Plate/metabolism , Neural Stem Cells/metabolism , Oligonucleotide Array Sequence Analysis , SOXB1 Transcription Factors/metabolism , Transcription Factors/deficiency , Transcription Factors/genetics , Transcriptional Activation , Xenopus , p300-CBP Transcription Factors/metabolism
4.
J Immunol ; 190(7): 3309-18, 2013 Apr 01.
Article in English | MEDLINE | ID: mdl-23460741

ABSTRACT

Peripheral lymphoid tissues, such as lymph nodes and Peyer's patches (PPs), are organs required for mounting highly efficient immune responses to small quantities of Ag. The compartmentalization of the cellular components involved in the immune response into distinct zones supports the function of these tissues; however, little is known about how this compartmentalization is achieved. In this study, we analyzed neonatal PP development and present evidence that the CD3(-)IL-7Rα(+) PP inducer cells that initially play a pivotal role in the formation of the PP anlagen are involved in the formation of B and T cell zones in neonatal mice. PP inducer cells migrate between these zones by undergoing chemokine receptor switching.


Subject(s)
Peyer's Patches/cytology , Peyer's Patches/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , B-Lymphocytes/cytology , Cell Movement/immunology , Female , Male , Mice , Peyer's Patches/metabolism , Receptors, Chemokine/metabolism , Receptors, Interleukin-7/metabolism , T-Lymphocytes/cytology , T-Lymphocytes, Helper-Inducer/metabolism , Time Factors
5.
Nature ; 457(7231): 896-900, 2009 Feb 12.
Article in English | MEDLINE | ID: mdl-19212410

ABSTRACT

Despite decades of research, the identity of the cells generating the first haematopoietic cells in mammalian embryos is unknown. Indeed, whether blood cells arise from mesodermal cells, mesenchymal progenitors, bipotent endothelial-haematopoietic precursors or haemogenic endothelial cells remains controversial. Proximity of endothelial and blood cells at sites of embryonic haematopoiesis, as well as their similar gene expression, led to the hypothesis of the endothelium generating blood. However, owing to lacking technology it has been impossible to observe blood cell emergence continuously at the single-cell level, and the postulated existence of haemogenic endothelial cells remains disputed. Here, using new imaging and cell-tracking methods, we show that embryonic endothelial cells can be haemogenic. By continuous long-term single-cell observation of mouse mesodermal cells generating endothelial cell and blood colonies, it was possible to detect haemogenic endothelial cells giving rise to blood cells. Living endothelial and haematopoietic cells were identified by simultaneous detection of morphology and multiple molecular and functional markers. Detachment of nascent blood cells from endothelium is not directly linked to asymmetric cell division, and haemogenic endothelial cells are specified from cells already expressing endothelial markers. These results improve our understanding of the developmental origin of mammalian blood and the potential generation of haematopoietic stem cells from embryonic stem cells.


Subject(s)
Blood Cells/cytology , Cell Differentiation , Hemangioblasts/cytology , Image Processing, Computer-Assisted , Video Recording , Animals , Cell Line , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryonic Stem Cells/cytology , Mesoderm/cytology , Mice , Microscopy, Fluorescence
6.
Proc Natl Acad Sci U S A ; 109(12): 4515-20, 2012 Mar 20.
Article in English | MEDLINE | ID: mdl-22392989

ABSTRACT

Several lines of evidence suggest that the adult hematopoietic system has multiple developmental origins, but the ontogenic relationship between nascent hematopoietic populations under this scheme is poorly understood. In an alternative theory, the earliest definitive blood precursors arise from a single anatomical location, which constitutes the cellular source for subsequent hematopoietic populations. To deconvolute hematopoietic ontogeny, we designed an embryo-rescue system in which the key hematopoietic factor Runx1 is reactivated in Runx1-null conceptuses at specific developmental stages. Using complementary in vivo and ex vivo approaches, we provide evidence that definitive hematopoiesis and adult-type hematopoietic stem cells originate predominantly in the nascent extraembryonic mesoderm. Our data also suggest that other anatomical sites that have been proposed to be sources of the definitive hematopoietic hierarchy are unlikely to play a substantial role in de novo blood generation.


Subject(s)
Hematopoietic Stem Cells/cytology , Alleles , Animals , Cell Lineage , Core Binding Factor Alpha 2 Subunit , Female , Flow Cytometry/methods , Gene Expression Regulation, Developmental , Hematopoiesis , In Situ Hybridization , Mice , Mice, Inbred C57BL , Models, Genetic , Time Factors , beta-Galactosidase/metabolism
7.
Development ; 138(24): 5357-68, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22071109

ABSTRACT

The mouse Flk1 gene is expressed in various mesodermal progenitor cells of developing embryos. Recent studies have shown that Flk1 expression marks multipotent mesodermal progenitors, giving rise to various hemato-cardiovascular cell lineages during development. Flk1 expression also marks hemato-cardiovascular cell lineages in differentiating embryonic stem (ES) cells, which may be used in transplantation decisions to treat cardiovascular diseases. Despite its developmental and clinical importance in cardiovascular tissues, the transcriptional regulatory system of Flk1 has remained unclear. Here, we report a novel enhancer of the mouse Flk1 gene directing early mesodermal expression during development as well as ES differentiation. The enhancer enriches various mesodermal progenitors, such as primitive erythropoietic progenitors, hemangioblast (BL-CFC) and cardiovascular progenitors (CV-CFC). The enhancer is activated by Bmp, Wnt and Fgf, and it contains Gata-, Cdx-, Tcf/Lef-, ER71/Etv2- and Fox-binding sites, some of which are bound specifically by each of these transcription factors. As these transcription factors are known to act under the control of the Bmp, Wnt and Fgf families, early Flk1 expression may be induced by cooperative interactions between Gata, Tcf/Lef, Cdx and ER71/Etv2 under the control of Bmp, Wnt and Fgf signaling. The enhancer is required for early Flk1 expression and for hemangioblast development during ES differentiation.


Subject(s)
Cardiovascular System/enzymology , Cardiovascular System/growth & development , Embryonic Stem Cells/enzymology , Hematopoietic Stem Cells/enzymology , Vascular Endothelial Growth Factor Receptor-2/metabolism , Animals , Cell Differentiation , Cells, Cultured , Enhancer Elements, Genetic , Female , Gene Expression Regulation, Developmental , Mesoderm/enzymology , Mice , Mice, Transgenic , Transcription Factors/metabolism , Vascular Endothelial Growth Factor Receptor-2/genetics
8.
Genes Cells ; 18(8): 704-21, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23795570

ABSTRACT

Etv2 is a critical determinant for the commitment of endothelial (EC) and hematopoietic (HPC) cells from mesoderm. Etv2 is assumed to be transiently required for EC commitment but dispensable after most ECs differentiate around E9.5. To confirm the time window of Etv2 requirement, Etv2 was ablated at different time points using ROSA26CreER mice. Unexpectedly, Etv2 ablation at E9.5 caused vascular remodeling defects in cranial and yolk sac vasculature. Immunostaining showed that Etv2+/VE-cadherin (VECAD)- cells were present around forming vasculature, mostly co-expressing Flk-1 with a small number of Etv2+/VECAD+ cells, indicating that Etv2+/Flk-1+/VECAD- cells are the major Etv2+ population promoting vascular remodeling around E9.5. Gene expression analysis showed up-regulation of Fgf proteins, Il-6, Glypican-3 and matrix metalloproteases in Etv2+/VEDAC- cells over Etv2-/VECAD+ mature ECs. Blockade of those factors caused reduced EC sprouting in ex vivo explant culture from E9.5 embryos, suggesting the functional significance of environmental factors derived from Etv2+ cells. Altogether, we propose that Etv2+/VEDAC- cells around E9.5-E10.5 provide extracellular factors to complete vascular morphogenesis in addition to becoming differentiated ECs incorporated into vessels. This insight for the new role of Ets protein in perivascular Flk-1+/VECAD-/(Etv2+) cells to induce expression of angiogenic factors may provide another strategy to control angiogenesis.


Subject(s)
Endothelial Cells/metabolism , Endothelium, Vascular/embryology , Morphogenesis , Transcription Factors/metabolism , Animals , Cell Differentiation , Cells, Cultured , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Endothelial Cells/cytology , Endothelium, Vascular/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental , Mesoderm/cytology , Mice , Transcription Factors/genetics
9.
Proc Natl Acad Sci U S A ; 108(34): 14234-9, 2011 Aug 23.
Article in English | MEDLINE | ID: mdl-21821793

ABSTRACT

After the first report of induced pluripotent stem cells (iPSCs), considerable efforts have been made to develop more efficient methods for generating iPSCs without foreign gene insertions. Here we show that Sendai virus vector, an RNA virus vector that carries no risk of integrating into the host genome, is a practical solution for the efficient generation of safer iPSCs. We improved the Sendai virus vectors by introducing temperature-sensitive mutations so that the vectors could be easily removed at nonpermissive temperatures. Using these vectors enabled the efficient production of viral/factor-free iPSCs from both human fibroblasts and CD34(+) cord blood cells. Temperature-shift treatment was more effective in eliminating remaining viral vector-related genes. The resulting iPSCs expressed human embryonic stem cell markers and exhibited pluripotency. We suggest that generation of transgene-free iPSCs from cord blood cells should be an important step in providing allogeneic iPSC-derived therapy in the future.


Subject(s)
Genetic Vectors/genetics , Induced Pluripotent Stem Cells/metabolism , Sendai virus/genetics , Temperature , Transgenes/genetics , Animals , Biomarkers/metabolism , Cell Differentiation , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Epigenesis, Genetic , Fetal Blood/cytology , Fibroblasts/metabolism , Germ Layers/cytology , Germ Layers/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Mice
10.
Dev Dyn ; 242(3): 254-68, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23335233

ABSTRACT

BACKGROUND: Early mesoderm can be classified into Flk-1+ or PDGF receptor alpha (PDGFRα)+ population, grossly representing lateral and paraxial mesoderm, respectively. It has been demonstrated that all endothelial (EC) and hematopoietic (HPC) cells are derived from Flk-1+ cells. Although PDGFRα+ cells give rise to ECs/HPCs in in vitro ES differentiation, whether PDGFRα+ population can become hemato-endothelial lineages has not been proved in mouse embryos. RESULTS: Using PDGFRαMerCreMer mice, PDGFRα+ early mesoderm was shown to contribute to endothelial cells including hemogenic ECs, fetal liver B lymphocytes, and Lin-Kit+Sca-1+ (KSL) cells. Contribution of PDGFRα+ mesoderm into ECs and HPCs was limited until E8.5, indicating that PDGFRα+/Flk-1+ population that exists until E8.5 may be the source for hemato-endothelial lineages from PDGFRα+ population. The functional significance of PDGFRα+ mesoderm in vascular development and hematopoiesis was confirmed by genetic deletion of Etv2 or restoration of Runx1 in PDGFRα+ cells. Etv2 deletion and Runx1 restoration in PDGFRα+ cells resulted in abnormal vascular remodeling and rescue of fetal liver CD45+ and Lin-Kit+Sca-1+ (KSL) cells, respectively. CONCLUSIONS: Endothelial and hematopoietic cells can be derived from PDGFRα+ early mesoderm in mice. PDGFRα+ mesoderm is functionally significant in vascular development and hematopoiesis from phenotype analysis of genetically modified embryos.


Subject(s)
Cell Lineage/physiology , Embryo, Mammalian/metabolism , Endothelial Cells/metabolism , Hematopoietic Stem Cells/metabolism , Mesoderm/embryology , Receptor, Platelet-Derived Growth Factor alpha/biosynthesis , Animals , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Embryo, Mammalian/cytology , Endothelial Cells/cytology , Hematopoietic Stem Cells/cytology , Liver/blood supply , Liver/cytology , Liver/embryology , Mesoderm/cytology , Mice , Mice, Transgenic , Receptor, Platelet-Derived Growth Factor alpha/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism
11.
J Biol Chem ; 287(33): 27983-96, 2012 Aug 10.
Article in English | MEDLINE | ID: mdl-22730381

ABSTRACT

The regulation of mesenchymal cell growth by signaling molecules plays an important role in maintaining tissue functions. Aberrant mesenchymal cell proliferation caused by disruption of this regulatory process leads to pathogenetic events such as fibrosis. In the current study we have identified a novel nuclear factor, Phf14, which controls the proliferation of mesenchymal cells by regulating PDGFRα expression. Phf14-null mice died just after birth due to respiratory failure. Histological analyses of the lungs of these mice showed interstitial hyperplasia with an increased number of PDGFRα(+) mesenchymal cells. PDGFRα expression was elevated in Phf14-null mesenchymal fibroblasts, resulting in increased proliferation. We demonstrated that Phf14 acts as a transcription factor that directly represses PDGFRα expression. Based on these results, we used an antibody against PDGFRα to successfully treat mouse lung fibrosis. This study shows that Phf14 acts as a negative regulator of PDGFRα expression in mesenchymal cells undergoing normal and abnormal proliferation, and is a potential target for new treatments of lung fibrosis.


Subject(s)
Cell Proliferation , Fibroblasts/metabolism , Gene Expression Regulation , Homeodomain Proteins/metabolism , Mesoderm/metabolism , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Repressor Proteins/metabolism , Transcription Factors/metabolism , Animals , Cell Line , Fibroblasts/pathology , Homeodomain Proteins/genetics , Mesoderm/pathology , Mice , Mice, Knockout , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/metabolism , Pulmonary Fibrosis/pathology , Pulmonary Fibrosis/therapy , Receptor, Platelet-Derived Growth Factor alpha/genetics , Repressor Proteins/genetics , Transcription Factors/genetics
12.
Blood ; 118(26): 6975-86, 2011 Dec 22.
Article in English | MEDLINE | ID: mdl-21911838

ABSTRACT

Etv2 (Ets Variant 2) has been shown to be an indispensable gene for the development of hematopoietic cells (HPCs)/endothelial cells (ECs). However, how Etv2 specifies the mesoderm-generating HPCs/ECs remains incompletely understood. In embryonic stem cell (ESC) differentiation culture and Etv2-null embryos, we show that Etv2 is dispensable for generating primitive Flk-1(+)/PDGFRα(+) mesoderm but is required for the progression of Flk-1(+)/PDGFRα(+) cells into vascular/hematopoietic mesoderm. Etv2-null ESCs and embryonic cells were arrested as Flk-1(+)/PDGFRα(+) and failed to generate Flk-1(+)/PDGFRα(-) mesoderm. Flk-1(+)/Etv2(+) early embryonic cells showed significantly higher hemato-endothelial potential than the Flk-1(+)/Etv2(-) population, suggesting that Etv2 specifies a hemato-endothelial subset of Flk-1(+) mesoderm. Critical hemato-endothelial genes were severely down-regulated in Etv2-null Flk-1(+) cells. Among those genes Scl, Fli1, and GATA2 were expressed simultaneously with Etv2 in early embryos and seemed to be critical targets. Etv2 reexpression in Etv2-null cells restored the development of CD41(+), CD45(+), and VE-cadherin(+) cells. Expression of Scl or Fli1 alone could also restore HPCs/ECs in the Etv2-null background, indicating that these 2 genes are critical downstream targets. Furthermore, VEGF induced Etv2 potently and rapidly in Flk-1(+) mesoderm. We propose that Flk-1(+)/PDGFRα(+) primitive mesoderm is committed into Flk-1(+)/PDGFRα(-) vascular mesoderm through Etv2 and that up-regulation of Etv2 by VEGF promotes this commitment.


Subject(s)
Mesoderm/metabolism , Receptor, Platelet-Derived Growth Factor alpha/genetics , Transcription Factors/genetics , Vascular Endothelial Growth Factor Receptor-2/genetics , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Cadherins/genetics , Cadherins/metabolism , Cell Differentiation/genetics , Cell Line , Cells, Cultured , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/embryology , Endothelium, Vascular/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Hematopoietic System/cytology , Hematopoietic System/embryology , Hematopoietic System/metabolism , In Situ Hybridization , Leukocyte Common Antigens/genetics , Leukocyte Common Antigens/metabolism , Mesoderm/embryology , Mice , Mice, Knockout , Mice, Transgenic , Microscopy, Fluorescence , Oligonucleotide Array Sequence Analysis , Platelet Membrane Glycoprotein IIb/genetics , Platelet Membrane Glycoprotein IIb/metabolism , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Transcription Factors/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism
13.
Stem Cells ; 30(4): 687-96, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22267325

ABSTRACT

Ets family protein Etv2 (also called ER71 or Etsrp) is a key factor for initiation of vascular and blood development from mesodermal cells. However, regulatory mechanisms and inducing signals for Etv2 expression have been largely unknown. Previously, we revealed that cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling enhanced differentiation of vascular progenitors into endothelial cells (ECs) and hematopoietic cells (HPCs) using an embryonic stem cell (ESC) differentiation system. Here, we show that PKA activation in an earlier differentiation stage can trigger EC/HPC differentiation through Etv2 induction. We found Etv2 was markedly upregulated by PKA activation preceding EC and HPC differentiation. We identified two cAMP response element (CRE) sequences in the Etv2 promoter and 5'-untranslated region and confirmed that CRE-binding protein (CREB) directly binds to the CRE sites and activates Etv2 transcription. Expression of a dominant negative form of CREB completely inhibited PKA-elicited Etv2 expression and induction of EC/HPCs from ESCs. Furthermore, blockade of PKA significantly inhibited Etv2 expression in ex vivo whole-embryo culture using Etv2-Venus knockin mice. These data indicated that PKA/CREB pathway is a critical regulator for the initiation of EC/HPC differentiation via Etv2 transcription. This early-stage molecular linkage between a triggering signal and transcriptional cascades for differentiation would provide novel insights in vascular and blood development and cell fate determination.


Subject(s)
Cell Differentiation , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Endothelial Cells/cytology , Hematopoietic Stem Cells/cytology , Proto-Oncogene Protein c-ets-1/metabolism , Signal Transduction , 5' Untranslated Regions/genetics , Animals , Base Sequence , Cell Line , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/enzymology , Endothelial Cells/metabolism , Enzyme Activation , Hematopoietic Stem Cells/metabolism , Mice , Models, Biological , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Proto-Oncogene Protein c-ets-1/genetics , RNA, Small Interfering/metabolism , Tissue Culture Techniques , Transcription, Genetic
14.
Protein Expr Purif ; 90(1): 20-6, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23628981

ABSTRACT

Leukemia inhibitor factor (LIF) is a three disulfide bridge-containing cytokine with numerous regulatory effects. In this report, we present the high level expression of a soluble recombinant human LIF (rhLIF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hLIF cDNA was cloned into pET3b vector, and transformed into E. coli OrigamiB(DE3) harboring the bacterial thioredoxin coexpression vector. By using an enzyme-based glucose release system (EnBase®) and high-aeration shake flask (Ultra Yield Flask™), the yield of soluble proteins was significantly improved in comparison to commonly-used 2 × YT media. The recombinant protein was purified via a single chromatographic step using an affinity tag-based protein purification system that processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Soluble rhLIF yield was estimated to be approximately 1mg/g of wet weight cells, with above 98% purity. The rhLIF protein specifically inhibited the proliferation of the murine myeloblastic leukemia M1 cell in a dose-dependent manner, and induced Stat3 phosphorylation in mouse ES cells. This novel expression and purification protocol for the production of recombinant hLIF is a simple, suitable, and effective method.


Subject(s)
Escherichia coli/genetics , Leukemia Inhibitory Factor/genetics , Leukemia Inhibitory Factor/isolation & purification , Animals , Embryonic Stem Cells/metabolism , Escherichia coli/metabolism , Genetic Vectors , Humans , Leukemia Inhibitory Factor/metabolism , Mice , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , STAT3 Transcription Factor/metabolism , Tumor Cells, Cultured
15.
Nature ; 446(7139): 1056-61, 2007 Apr 26.
Article in English | MEDLINE | ID: mdl-17377529

ABSTRACT

The first haematopoietic stem cells (HSCs) appear in the aorta-gonad-mesonephros (AGM) region, major vitelline and umbilical vessels, and placenta; however, whether they arise locally or from immigrant yolk sac precursor cells remains unclear. This issue is best addressed by measuring cell-lineage relationships rather than cell potentials. To undertake long-term in vivo tracing of yolk sac cells, we designed a non-invasive pulse-labelling system based on Cre/loxP recombination. Here we show that in Runx1(+/-) (runt-related transcription factor 1) heterozygous mice, yolk sac cells expressing Runx1 at embryonic day 7.5 develop into fetal lymphoid progenitors and adult HSCs. During mid-gestation the labelled (embryonic day 7.5) yolk sac cells colonize the umbilical cord, the AGM region and subsequently the embryonic liver. This raises the possibility that some HSCs associated with major embryonic vasculature are derived from yolk sac precursors. We observed virtually no contribution of the labelled cells towards the yolk sac vasculature, indicating early segregation of endothelial and haematopoietic lineages.


Subject(s)
Cell Lineage , Hematopoiesis , Hematopoietic Stem Cells/cytology , Yolk Sac/cytology , Animals , Aorta , Core Binding Factor Alpha 2 Subunit/analysis , Core Binding Factor Alpha 2 Subunit/deficiency , Core Binding Factor Alpha 2 Subunit/genetics , Female , Fetus/cytology , Fetus/immunology , Gene Expression Regulation, Developmental , Genes, Reporter/genetics , Hematopoietic Stem Cells/metabolism , Heterozygote , Lymphocytes/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Time Factors , Umbilical Arteries , Umbilical Veins , Yolk Sac/metabolism
16.
Blood ; 114(20): 4383-92, 2009 Nov 12.
Article in English | MEDLINE | ID: mdl-19770359

ABSTRACT

Hematopoietic stem cell (HSC) proliferation is tightly regulated by a poorly understood complex of positive and negative cell-cycle regulatory mechanisms. Necdin (Ndn) is an evolutionally conserved multifunctional protein that has been implicated in cell-cycle regulation of neuronal cells. Here, we provide evidence that necdin plays an important role in restricting excessive HSC proliferation during hematopoietic regeneration. We identify Ndn as being preferentially expressed in the HSC population on the basis of gene expression profiling and demonstrate that mice deficient in Ndn show accelerated recovery of the hematopoietic system after myelosuppressive injury, whereas no overt abnormality is seen in steady-state hematopoiesis. In parallel, after myelosuppression, Ndn-deficient mice exhibit an enhanced number of proliferating HSCs. Based on these findings, we propose that necdin functions in a negative feedback loop that prevents excessive proliferation of HSCs during hematopoietic regeneration. These data suggest that the inhibition of necdin after clinical myelosuppressive treatment (eg, chemotherapy, HSC transplantation) may provide therapeutic benefits by accelerating hematologic recovery.


Subject(s)
Hematopoiesis/physiology , Hematopoietic Stem Cells/physiology , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Regeneration/genetics , Animals , Apoptosis , Cell Proliferation , Gene Expression , Gene Expression Profiling , Immunohistochemistry , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction
17.
Stem Cells ; 28(9): 1571-80, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20641035

ABSTRACT

Adult stem cells, which are characterized by their capacity for self-renewal and differentiation, participate in tissue homeostasis and response to injury. They are thought to enter a state of relative quiescence, known as reversible cell cycle arrest, but the underlying molecular mechanisms remain poorly characterized. Previous data from our laboratory has shown that housekeeping gene expression is downregulated in melanocyte stem cells (MelSCs), suggesting a global suppression of mRNA transcription. We now show, using antibodies against specific phosphorylated forms of RNA polymerase II (RNApII), that adult MelSCs do not undergo productive mRNA transcription elongation, while RNApII is activated and initialized, ready to synthesize mRNA upon stimulation, and that the RNApII kinase CDK9 is absent in adult MelSCs. Interestingly, other adult stem cells also, including keratinocyte, muscle, spermatogonia, and hematopoietic stem cells, showed a similar absence of RNApII phosphorylation. Although it is difficult to show the functional significance of this observation in vivo, CDK9 inhibition resulted in enhanced survival of cells that are deprived from survival factors. We conclude that the absence of productive mRNA transcription is an early, specific, and conserved characteristic of adult stem cells. Downregulation of mRNA transcription may lead to decreased rates of metabolism, and protection from cellular and genetic damage. Screening heterogeneous tissues, including tumors, for transcriptionally quiescent cells may result in the identification of cells with stem cell-like phenotypes.


Subject(s)
Adult Stem Cells/enzymology , Cellular Senescence , Melanocytes/enzymology , Protein Processing, Post-Translational , RNA Polymerase II/metabolism , Animals , Cell Differentiation , Cell Proliferation , Cellular Senescence/genetics , Cyclin-Dependent Kinase 9/genetics , Cyclin-Dependent Kinase 9/metabolism , Down-Regulation , Genotype , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Transgenic , Mutation , NIH 3T3 Cells , Phenotype , Phosphorylation , Promoter Regions, Genetic , Proteins/genetics , Proteins/metabolism , Proto-Oncogene Proteins c-kit/metabolism , RNA, Untranslated , Serine , Signal Transduction , Stem Cell Factor/metabolism , Time Factors , Transcription, Genetic , Transfection
18.
Stem Cells ; 28(2): 365-75, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19859984

ABSTRACT

Lnk is an intracellular adaptor protein reported as a negative regulator of proliferation in c-Kit positive, Sca-1 positive, lineage marker-negative (KSL) bone marrow cells. The KSL fraction in mouse bone marrow is believed to represent a population of hematopoietic and endothelial progenitor cells (EPCs). We report here that, in vitro, Lnk(-/-) KSL cells form more EPC colonies than Lnk(+/+) KSL cells and show higher expression levels of endothelial marker genes, including CD105, CD144, Tie-1, and Tie2, than their wild-type counterparts. In vivo, the administration of Lnk(+/+) KSL cells to a mouse spinal cord injury model promoted angiogenesis, astrogliosis, axon growth, and functional recovery following injury, with Lnk(-/-) KSL being significantly more effective in inducing and promoting these regenerative events. At day 3 following injury, large vessels could be observed in spinal cords treated with KSL cells, and reactive astrocytes were found to have migrated along these large vessels. We could further show that the enhancement of astrogliosis appears to be caused in conjunction with the acceleration of angiogenesis. These findings suggest that Lnk deletion reinforces the commitment of KSL cells to EPCs, promoting subsequent repair of injured spinal cord through the acceleration of angiogenesis and astrogliosis.


Subject(s)
Astrocytes/physiology , Bone Marrow Cells/cytology , Hematopoietic Stem Cells/physiology , Neovascularization, Physiologic/physiology , Proteins/physiology , Spinal Cord Injuries/physiopathology , Adaptor Proteins, Signal Transducing , Animals , Astrocytes/cytology , Astrocytes/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Immunohistochemistry , Intracellular Signaling Peptides and Proteins , Membrane Proteins , Mice , Mice, Knockout , Neovascularization, Physiologic/genetics , Proteins/genetics , Spinal Cord Injuries/metabolism
19.
J Cell Biol ; 173(3): 333-9, 2006 May 08.
Article in English | MEDLINE | ID: mdl-16651378

ABSTRACT

Melanoblasts (Mbs) are thought to be strictly regulated by cell-cell interactions with epidermal keratinocytes, although the precise molecular mechanism of the regulation has been elusive. Notch signaling, whose activation is mediated by cell-cell interactions, is implicated in a broad range of developmental processes. We demonstrate the vital role of Notch signaling in the maintenance of Mbs, as well as melanocyte stem cells (MSCs). Conditional ablation of Notch signaling in the melanocyte lineage leads to a severe defect in hair pigmentation, followed by intensive hair graying. The defect is caused by a dramatic elimination of Mbs and MSCs. Furthermore, targeted overexpression of Hes1 is sufficient to protect Mbs from the elimination by apoptosis. Thus, these data provide evidence that Notch signaling, acting through Hes1, plays a crucial role in the survival of immature Mbs by preventing initiation of apoptosis.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/physiology , Homeodomain Proteins/physiology , Melanocytes/cytology , Receptors, Notch/physiology , Signal Transduction/physiology , Stem Cells/cytology , Animals , Apoptosis/drug effects , Apoptosis/physiology , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Cycle Proteins/genetics , Cell Survival/drug effects , Cell Survival/physiology , Dipeptides/pharmacology , Enzyme Inhibitors/pharmacology , Epidermal Cells , Epidermis/embryology , Epidermis/metabolism , Gene Expression/genetics , Hair Color/genetics , Homeodomain Proteins/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Intramolecular Oxidoreductases/metabolism , Jagged-2 Protein , Melanocytes/metabolism , Membrane Proteins/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Mice, Transgenic , Receptor, Notch1/metabolism , Stem Cells/metabolism , Transcription Factor HES-1
20.
Mol Cell Neurosci ; 45(2): 121-31, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20599619

ABSTRACT

The orphan nuclear receptor TLX has been proposed to act as a repressor of cell cycle inhibitors to maintain the neural stem cells in an undifferentiated state, and prevents commitment into astrocyte lineages. However, little is known about the mechanism of TLX in neuronal lineage commitment and differentiation. A majority of adult rat hippocampus-derived progenitors (AHPs) cultured in the presence of FGF express a high level of TLX and a fraction of these cells also express the proneural gene MASH1. Upon FGF withdrawal, TLX rapidly decreased, while MASH1 was intensely expressed within 1h, decreasing gradually to disappear at 24h. Adenoviral transduction of TLX in AHP cells in the absence of FGF transiently increased cell proliferation, however, later resulted in neuronal differentiation by inducing MASH1, Neurogenin1, DCX, and MAP2ab. Furthermore, TLX directly targets and activates the MASH1 promoter through interaction with Sp1, recruiting co-activators whereas dismissing the co-repressor HDAC4. Conversely, silencing of TLX in AHPs decreased beta-III tubulin and DCX expression and promoted glial differentiation. Our results thus suggest that TLX not only acts as a repressor of cell cycle and glial differentiation but also activates neuronal lineage commitment in AHPs.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Hippocampus/growth & development , Neurogenesis/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Repressor Proteins/metabolism , Transcriptional Activation , Adenoviridae , Animals , Basic Helix-Loop-Helix Transcription Factors/analysis , Cell Lineage/genetics , Cells, Cultured , Doublecortin Domain Proteins , Doublecortin Protein , Fibroblast Growth Factors/metabolism , Fibroblast Growth Factors/pharmacology , Histone Deacetylases/analysis , Humans , Microtubule-Associated Proteins/analysis , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/physiology , Neuropeptides/analysis , Promoter Regions, Genetic , Rats , Receptors, Cytoplasmic and Nuclear/genetics , Repressor Proteins/genetics , Sp1 Transcription Factor/analysis , Sp1 Transcription Factor/metabolism , Tubulin/analysis
SELECTION OF CITATIONS
SEARCH DETAIL