Publications by authors named "Giovanni Amabile"

20 Publications

  • Page 1 of 1

Decellularized Extracellular Matrices and Cardiac Differentiation: Study on Human Amniotic Fluid-Stem Cells.

Int J Mol Sci 2020 Aug 31;21(17). Epub 2020 Aug 31.

Haman Anatomy and Cell Differentiation Lab, Department of Medicine and Aging Sciences, University "G.d'Annunzio" of Chieti-Pescara, 66100 Chieti, Italy.

Cell therapy with a variety of stem populations is increasingly being investigated as a promising regenerative strategy for cardiovascular (CV) diseases. Their combination with adequate scaffolds represents an improved therapeutic approach. Recently, several biomaterials were investigated as scaffolds for CV tissue repair, with decellularized extracellular matrices (dECMs) arousing increasing interest for cardiac tissue engineering applications. The aim of this study was to analyze whether dECMs support the cardiac differentiation of AF stem cells. These perinatal stem cells, which can be easily isolated without ethical or safety limitations, display a high cardiac differentiative potential. Differentiation was previously achieved by culturing them on Matrigel, but this 3D scaffold is not transplantable. The identification of a new transplantable scaffold able to support AF stem cell cardiac differentiation is pivotal prior to encouraging translation of in vitro studies in animal model preclinical investigations. Our data demonstrated that decellularized extracellular matrices already used in cardiac surgery (the porcine CorPATCH and the equine MatrixPatch) can efficiently support the proliferation and cardiac differentiation of AF stem cells and represent a useful cellular scaffold to be transplanted with stem cells in animal hosts.
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http://dx.doi.org/10.3390/ijms21176317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504221PMC
August 2020

Epigenetic Features of Human Perinatal Stem Cells Redefine Their Stemness Potential.

Cells 2020 05 24;9(5). Epub 2020 May 24.

Department of Medicine and Aging Sciences, "G. D'Annunzio" University of Chieti- Pescara, 66100 Chieti, Italy.

Human perinatal stem cells (SCs) can be isolated from fetal annexes without ethical or safety limitations. They are generally considered multipotent; nevertheless, their biological characteristics are still not fully understood. The aim of this study was to investigate the pluripotency potential of human perinatal SCs as compared to human induced pluripotent stem cells (hiPSCs). Despite the low expression of the pluripotent factors NANOG, OCT4, SOX2, and C-KIT in perinatal SC, we observed minor differences in the promoters DNA-methylation profile of these genes with respect to hiPSCs; we also demonstrated that in perinatal SCs miR-145-5p had an inverse trend in comparison to these stemness markers, suggesting that NANOG, OCT4, and SOX2 were regulated at the post-transcriptional level. The reduced expression of stemness markers was also associated with shorter telomere lengths and shift of the oxidative metabolism between hiPSCs and fetal annex-derived cells. Our findings indicate the differentiation ability of perinatal SCs might not be restricted to the mesenchymal lineage due to an epigenetic barrier, but other regulatory mechanisms such as telomere shortening or metabolic changes might impair their differentiation potential and challenge their clinical application.
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http://dx.doi.org/10.3390/cells9051304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290760PMC
May 2020

Induced Pluripotent Stem Cell-Derived Red Blood Cells and Platelet Concentrates: From Bench to Bedside.

Cells 2017 Dec 27;7(1). Epub 2017 Dec 27.

ADIENNE, via Zurigo 46, 6900 Lugano, Switzerland.

Red blood cells and platelets are anucleate blood components indispensable for oxygen delivery and hemostasis, respectively. Derivation of these blood elements from induced pluripotent stem (iPS) cells has the potential to develop blood donor-independent and genetic manipulation-prone products to complement or replace current transfusion banking, also minimizing the risk of alloimmunization. While the production of erythrocytes from iPS cells has challenges to overcome, such as differentiation into adult-type phenotype that functions properly after transfusion, platelet products are qualitatively and quantitatively approaching a clinically-applicable level owing to advances in expandable megakaryocyte (MK) lines, platelet-producing bioreactors, and novel reagents. Guidelines that assure the quality of iPS cells-derived blood products for clinical application represent a novel challenge for regulatory agencies. Considering the minimal risk of tumorigenicity and the expected significant demand of such products, ex vivo production of iPS-derived blood components can pave the way for iPS translation into the clinic.
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http://dx.doi.org/10.3390/cells7010002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789275PMC
December 2017

The second hit of DNA methylation.

Mol Cell Oncol 2016 May 29;3(3):e1093690. Epub 2015 Oct 29.

Harvard Medical School, Boston, MA, USA; Harvard Stem Cell Institute, Boston, MA, USA; ADIENNE, Lugano, Switzerland.

Gene expression programs are tightly regulated by heritable "epigenetic" information, which is stored as chemical modifications of histones and DNA. With the recent development of sequencing-based epigenome mapping technologies and cancer cellular reprogramming, the tools are now in hand to analyze the epigenetic contribution to human cancer.
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http://dx.doi.org/10.1080/23723556.2015.1093690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4909463PMC
May 2016

SALL4, the missing link between stem cells, development and cancer.

Gene 2016 Jun 16;584(2):111-9. Epub 2016 Feb 16.

Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, New Research Building Room 652D, Boston, MA 02115, USA. Electronic address:

There is a growing body of evidence supporting that cancer cells share many similarities with embryonic stem cells (ESCs). For example, aggressive cancers and ESCs share a common gene expression signature that includes hundreds of genes. Since ESC genes are not present in most adult tissues, they could be ideal candidate targets for cancer-specific diagnosis and treatment. This is an exciting cancer-targeting model. The major hurdle to test this model is to identify the key factors/pathway(s) within ESCs that are responsible for the cancer phenotype. SALL4 is one of few genes that can establish this link. The first publication of SALL4 is on its mutation in a human inherited disorder with multiple developmental defects. Since then, over 300 papers have been published on various aspects of this gene in stem cells, development, and cancers. This review aims to summarize our current knowledge of SALL4, including a SALL4-based approach to classify and target cancers. Many questions about this important gene still remain unanswered, specifically, on how this gene regulates cell fates at a molecular level. Understanding SALL4's molecular functions will allow development of specific targeted approaches in the future.
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http://dx.doi.org/10.1016/j.gene.2016.02.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4823161PMC
June 2016

Dissecting the role of aberrant DNA methylation in human leukaemia.

Nat Commun 2015 May 22;6:7091. Epub 2015 May 22.

1] Harvard Medical School, Boston, Massachusetts 02115, USA [2] Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts 02138, USA [3] Cancer Science Institute, National University of Singapore, Singapore 66123, Singapore.

Chronic myeloid leukaemia (CML) is a myeloproliferative disorder characterized by the genetic translocation t(9;22)(q34;q11.2) encoding for the BCR-ABL fusion oncogene. However, many molecular mechanisms of the disease progression still remain poorly understood. A growing body of evidence suggests that the epigenetic abnormalities are involved in tyrosine kinase resistance in CML, leading to leukaemic clone escape and disease propagation. Here we show that, by applying cellular reprogramming to primary CML cells, aberrant DNA methylation contributes to the disease evolution. Importantly, using a BCR-ABL inducible murine model, we demonstrate that a single oncogenic lesion triggers DNA methylation changes, which in turn act as a precipitating event in leukaemia progression.
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http://dx.doi.org/10.1038/ncomms8091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4443494PMC
May 2015

Treatment of chronic myelogenous leukemia by blocking cytokine alterations found in normal stem and progenitor cells.

Cancer Cell 2015 May;27(5):671-81

Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA; Cancer Science Institute, National University of Singapore, Singapore 119077, Singapore. Electronic address:

Leukemic cells disrupt normal patterns of blood cell formation, but little is understood about the mechanism. We investigated whether leukemic cells alter functions of normal hematopoietic stem and progenitor cells. Exposure to chronic myelogenous leukemia (CML) caused normal mouse hematopoietic progenitor cells to divide more readily, altered their differentiation, and reduced their reconstitution and self-renewal potential. Interestingly, the normal bystander cells acquired gene expression patterns resembling their malignant counterparts. Therefore, much of the leukemia signature is mediated by extrinsic factors. Indeed, IL-6 was responsible for most of these changes. Compatible results were obtained when human CML were cultured with normal human hematopoietic progenitor cells. Furthermore, neutralization of IL-6 prevented these changes and treated the disease.
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http://dx.doi.org/10.1016/j.ccell.2015.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4447336PMC
May 2015

Notch inhibition allows oncogene-independent generation of iPS cells.

Nat Chem Biol 2014 Aug 22;10(8):632-639. Epub 2014 Jun 22.

Harvard Stem Cell Institute, Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.

The reprogramming of somatic cells to pluripotency using defined transcription factors holds great promise for biomedicine. However, human reprogramming remains inefficient and relies either on the use of the potentially dangerous oncogenes KLF4 and CMYC or the genetic inhibition of the tumor suppressor gene p53. We hypothesized that inhibition of signal transduction pathways that promote differentiation of the target somatic cells during development might relieve the requirement for non-core pluripotency factors during induced pluripotent stem cell (iPSC) reprogramming. Here, we show that inhibition of Notch greatly improves the efficiency of iPSC generation from mouse and human keratinocytes by suppressing p21 in a p53-independent manner and thereby enriching for undifferentiated cells capable of long-term self-renewal. Pharmacological inhibition of Notch enabled routine production of human iPSCs without KLF4 and CMYC while leaving p53 activity intact. Thus, restricting the development of somatic cells by altering intercellular communication enables the production of safer human iPSCs.
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http://dx.doi.org/10.1038/nchembio.1552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4310751PMC
August 2014

Sox4 is a key oncogenic target in C/EBPα mutant acute myeloid leukemia.

Cancer Cell 2013 Nov 31;24(5):575-88. Epub 2013 Oct 31.

Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02215, USA; Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.

Mutation or epigenetic silencing of the transcription factor C/EBPα is observed in ∼10% of patients with acute myeloid leukemia (AML). In both cases, a common global gene expression profile is observed, but downstream targets relevant for leukemogenesis are not known. Here, we identify Sox4 as a direct target of C/EBPα whereby its expression is inversely correlated with C/EBPα activity. Downregulation of Sox4 abrogated increased self-renewal of leukemic cells and restored their differentiation. Gene expression profiles of leukemia-initiating cells (LICs) from both Sox4 overexpression and murine C/EBPα mutant AML models clustered together but differed from other types of AML. Our data demonstrate that Sox4 overexpression resulting from C/EBPα inactivation contributes to the development of leukemia with a distinct LIC phenotype.
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http://dx.doi.org/10.1016/j.ccr.2013.09.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4038627PMC
November 2013

DNMT1-interacting RNAs block gene-specific DNA methylation.

Nature 2013 Nov 9;503(7476):371-6. Epub 2013 Oct 9.

1] Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts 02115, USA [2] Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115, USA [3] Università Cattolica del Sacro Cuore, Institute of Hematology, L.go A. Gemelli 8, Rome 00168, Italy [4].

DNA methylation was first described almost a century ago; however, the rules governing its establishment and maintenance remain elusive. Here we present data demonstrating that active transcription regulates levels of genomic methylation. We identify a novel RNA arising from the CEBPA gene locus that is critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extend the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1-RNA interactions and suggest strategies for gene-selective demethylation of therapeutic targets in human diseases.
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http://dx.doi.org/10.1038/nature12598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3870304PMC
November 2013

C/EBPα is required for development of dendritic cell progenitors.

Blood 2013 May 1;121(20):4073-81. Epub 2013 Apr 1.

Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA.

Dendritic cells (DCs) are master regulators of the immune system, but molecular regulation of early DC differentiation has been poorly understood. Here, we report that the transcription factor C/EBPα coordinates the development of progenitor cells required for production of multiple categories of DCs. C/EBPα was needed for differentiation from stem/progenitor cells to common DC progenitors (CDPs), but not for transition of CDP to mature DCs. C/EBPα deletion in mature DCs did not affect their numbers or function, suggesting that this transcription factor is not needed for maintenance of DCs in lymphoid tissues. ChIP-seq and microarrays were used to identify candidate genes regulated by C/EBPα and required for DC formation. Genes previously shown to be critical for DC formation were bound by C/EBPα, and their expression was decreased in the earliest hematopoietic compartments in the absence of C/EBPα. These data indicate that C/EBPα is important for the earliest stages of steady-state DC differentiation.
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http://dx.doi.org/10.1182/blood-2012-10-463448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656447PMC
May 2013

C/EBPa controls acquisition and maintenance of adult haematopoietic stem cell quiescence.

Nat Cell Biol 2013 Apr 17;15(4):385-94. Epub 2013 Mar 17.

Harvard Stem Cell Institute, Harvard Medical School, and Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115, USA.

In blood, the transcription factor C/EBPa is essential for myeloid differentiation and has been implicated in regulating self-renewal of fetal liver haematopoietic stem cells (HSCs). However, its function in adult HSCs has remained unknown. Here, using an inducible knockout model we found that C/EBPa-deficient adult HSCs underwent a pronounced increase in number with enhanced proliferation, characteristics resembling fetal liver HSCs. Consistently, transcription profiling of C/EBPa-deficient HSCs revealed a gene expression program similar to fetal liver HSCs. Moreover, we observed that age-specific Cebpa expression correlated with its inhibitory effect on the HSC cell cycle. Mechanistically we identified N-Myc as a downstream target of C/EBPa, and loss of C/EBPa resulted in de-repression of N-Myc. Our data establish C/EBPa as a central determinant in the switch from fetal to adult HSCs.
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http://dx.doi.org/10.1038/ncb2698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3781213PMC
April 2013

In vivo generation of transplantable human hematopoietic cells from induced pluripotent stem cells.

Blood 2013 Feb 4;121(8):1255-64. Epub 2012 Dec 4.

Harvard Medical School, Boston, MA 02115, USA.

Lineage-restricted cells can be reprogrammed to a pluripotent state known as induced pluripotent stem (iPS) cells through overexpression of 4 transcription factors. iPS cells are similar to human embryonic stem (hES) cells and have the same ability to generate all the cells of the human body, including blood cells. However, this process is extremely inefficient and to date has been unsuccessful at differentiating iPS into hematopoietic stem cells (HSCs). We hypothesized that iPS cells, injected into NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ immunocompromised (NSG) mice could give rise to hematopoietic stem/progenitor cells (HSPCs) during teratoma formation. Here, we report a novel in vivo system in which human iPS cells differentiate within teratomas to derive functional myeloid and lymphoid cells. Similarly, HSPCs can be isolated from teratoma parenchyma and reconstitute a human immune system when transplanted into immunodeficient mice. Our data provide evidence that in vivo generation of patient customized cells is feasible, providing materials that could be useful for transplantation, human antibody generation, and drug screening applications.
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http://dx.doi.org/10.1182/blood-2012-06-434407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3701251PMC
February 2013

C/EBPγ deregulation results in differentiation arrest in acute myeloid leukemia.

J Clin Invest 2012 Dec 19;122(12):4490-504. Epub 2012 Nov 19.

Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts, USA.

C/EBPs are a family of transcription factors that regulate growth control and differentiation of various tissues. We found that C/EBPγ is highly upregulated in a subset of acute myeloid leukemia (AML) samples characterized by C/EBPα hypermethylation/silencing. Similarly, C/EBPγ was upregulated in murine hematopoietic stem/progenitor cells lacking C/EBPα, as C/EBPα mediates C/EBPγ suppression. Studies in myeloid cells demonstrated that CEBPG overexpression blocked neutrophilic differentiation. Further, downregulation of Cebpg in murine Cebpa-deficient stem/progenitor cells or in human CEBPA-silenced AML samples restored granulocytic differentiation. In addition, treatment of these leukemias with demethylating agents restored the C/EBPα-C/EBPγ balance and upregulated the expression of myeloid differentiation markers. Our results indicate that C/EBPγ mediates the myeloid differentiation arrest induced by C/EBPα deficiency and that targeting the C/EBPα-C/EBPγ axis rescues neutrophilic differentiation in this unique subset of AMLs.
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http://dx.doi.org/10.1172/JCI65102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3533560PMC
December 2012

RUNX1 regulates the CD34 gene in haematopoietic stem cells by mediating interactions with a distal regulatory element.

EMBO J 2011 Aug 26;30(19):4059-70. Epub 2011 Aug 26.

Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Center for Life Science, Boston, MA, USA.

The transcription factor RUNX1 is essential to establish the haematopoietic gene expression programme; however, the mechanism of how it activates transcription of haematopoietic stem cell (HSC) genes is still elusive. Here, we obtained novel insights into RUNX1 function by studying regulation of the human CD34 gene, which is expressed in HSCs. Using transgenic mice carrying human CD34 PAC constructs, we identified a novel downstream regulatory element (DRE), which is bound by RUNX1 and is necessary for human CD34 expression in long-term (LT)-HSCs. Conditional deletion of Runx1 in mice harbouring human CD34 promoter-DRE constructs abrogates human CD34 expression. We demonstrate by chromosome conformation capture assays in LT-HSCs that the DRE physically interacts with the human CD34 promoter. Targeted mutagenesis of RUNX binding sites leads to perturbation of this interaction and decreased human CD34 expression in LT-HSCs. Overall, our in vivo data provide novel evidence about the role of RUNX1 in mediating interactions between distal and proximal elements of the HSC gene CD34.
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http://dx.doi.org/10.1038/emboj.2011.285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209778PMC
August 2011

Induced pluripotent stem cells: current progress and potential for regenerative medicine.

Trends Mol Med 2009 Feb 21;15(2):59-68. Epub 2009 Jan 21.

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.

Lineage-restricted cells can be reprogrammed to a pluripotent state through overexpression of defined transcription factors. Here, we summarize recent progress in the direct reprogramming field and discuss data comparing embryonic stem (ES) and induced pluripotent stem (iPS) cells. Results from many independent groups suggest that mouse and human iPS cells, once established, generally exhibit a normal karyotype, are transcriptionally and epigenetically similar to ES cells and maintain the potential to differentiate into derivatives of all germ layers. Recent developments provide optimism that safe, viral-free human iPS cells could be derived routinely in the near future. An important next step will be to identify ways of assessing which iPS cell lines are sufficiently reprogrammed and safe to use for therapeutic applications. The approach of generating patient-specific pluripotent cells will undoubtedly transform regenerative medicine in many ways.
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http://dx.doi.org/10.1016/j.molmed.2008.12.003DOI Listing
February 2009

Reversine, a novel Aurora kinases inhibitor, inhibits colony formation of human acute myeloid leukemia cells.

Mol Cancer Ther 2008 May;7(5):1140-9

CEINGE, Biotecnologie Avanzate, Via Comunale Margherita 482, 80131 Naples, Italy.

The demonstration that the small synthetic molecule reversine [2-(4-morpholinoanilino)-N6-cyclohexyladenine] promotes the dedifferentiation of committed cells into multipotent progenitor-type cells has raised hopes on the exploitation of this small chemical tool for the generation of stem cells. Here, we show that reversine causes a failure in cytokinesis and induces polyploidization. These effects of reversine are due to the inhibition of Aurora A and B, two related kinases that are implicated in several aspects of mitosis and that are frequently amplified and overexpressed in human tumors. Reversine inhibits the phosphorylation of histone H3, a direct downstream target of Aurora kinases. Similarly to the Aurora kinase inhibitor VX-680, which has recently entered phase II clinical trials for cancer treatment, reversine inhibited colony formation of leukemic cells from patients with acute myeloid leukemia but was significantly less toxic than VX-680 on cells from healthy donors. The crystal structure of the reversine-Aurora B kinase complex shows that reversine is a novel class of ATP-competitive Aurora kinase inhibitors. Thus, although our studies raise serious doubts on the application of reversine in regenerative medicine, they support the paradigm that reversine might be a useful agent in cancer chemotherapy.
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http://dx.doi.org/10.1158/1535-7163.MCT-07-2051DOI Listing
May 2008

The hypomorphic Gata1low mutation alters the proliferation/differentiation potential of the common megakaryocytic-erythroid progenitor.

Blood 2007 Feb 12;109(4):1460-71. Epub 2006 Oct 12.

Department of Hematology, Oncology, and Molecular Medicine, Istituto Superiore Sanità, Rome, Italy.

Recent evidence suggests that mutations in the Gata1 gene may alter the proliferation/differentiation potential of hemopoietic progenitors. By single-cell cloning and sequential replating experiments of prospectively isolated progenitor cells, we demonstrate here that the hypomorphic Gata1low mutation increases the proliferation potential of a unique class of progenitor cells, similar in phenotype to adult common erythroid/megakaryocytic progenitors (MEPs), but with the "unique" capacity to generate erythroblasts, megakaryocytes, and mast cells in vitro. Conversely, progenitor cells phenotypically similar to mast cell progenitors (MCPs) are not detectable in the marrow from these mutants. At the single-cell level, about 11% of Gata1low progenitor cells, including MEPs, generate cells that will continue to proliferate in cultures for up to 4 months. In agreement with these results, trilineage (erythroid, megakaryocytic, and mastocytic) cell lines are consistently isolated from bone marrow and spleen cells of Gata1low mice. These results confirm the crucial role played by Gata1 in hematopoietic commitment and identify, as a new target for the Gata1 action, the restriction point at which common myeloid progenitors become either MEPs or MCPs.
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http://dx.doi.org/10.1182/blood-2006-07-030726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1794062PMC
February 2007

An autocrine loop involving ret and glial cell-derived neurotrophic factor mediates retinoic acid-induced neuroblastoma cell differentiation.

Mol Cancer Res 2006 Jul;4(7):481-8

Istituto per l'Endocrinologia e l'Oncologia Sperimentale del Consiglio Nazionale delle Ricerche G. Salvatore, Naples, Italy.

In several neuroblastoma cell lines, retinoic acid (RA)-induced differentiation is coupled to increased expression of functional neurotrophic factor receptors, including Trk family receptors and the glial cell-derived neurotrophic factor receptor, Ret. In several cases, increased expression is dependent on signaling through TrkB. Unlike TrkA and TrkB, Ret has never been implicated as a prognostic marker for neuroblastomas. SK-N-BE(2) cells do not express any of Trk family receptors; therefore, they are a choice system to study the specific role of Ret in RA-induced differentiation. Using a 2'-fluoro-RNA aptamer and a truncated Ret protein as specific inhibitors of Ret, we show that RA-induced differentiation is mediated by a positive autocrine loop that sustains Ret downstream signaling and depends on glial cell-derived neurotrophic factor expression and release. This report shows that in SK-N-BE(2) cells, stimulation of Ret is a major upstream mechanism needed to mediate RA-induced differentiation. These results provide important insights on the molecular mechanism of RA action, which might be relevant for the development of biologically based therapeutic strategies.
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http://dx.doi.org/10.1158/1541-7786.MCR-06-0050DOI Listing
July 2006

Isolation of TPO-dependent subclones from the multipotent 32D cell line.

Blood Cells Mol Dis 2005 Sep-Oct;35(2):241-52

Department of Cell Biology and Neurosciences, Istituto Superiore Sanità, Rome.

Using thrombopoietin (TPO), as selective pressure, several TPO-dependent clones were isolated from the murine multipotential IL-3-dependent cell line 32D. Four of them were fully characterized. They depended on TPO for survival and proliferation and, although retaining the capacity to grow in IL-3, did not respond to either EPO, G-CSF or GM-CSF. 32D TPO cells were heterogeneous in morphology and ranged from small cells, with a DNA content nearly tetraploid and a modal chromosome no. 66, to cells 50-75 microm in diameter containing multiple (up to 5-6) interconnected nuclei with a clear megakaryocyte (Mk) morphology by electron microscopy. Cell sorter isolation and single cell cloning experiments indicated that the small cells were those capable to proliferate in TPO and to generate the larger ones over time. 32D TPO cells expressed Mk-specific markers by FACS (CD41, CD61 and 2D5) and RT-PCR (acetyl cholinesterase E and platelet factor 4) and their unique profile, by gene array analysis, included expression of urokinase plasminogen activator surface receptor (CD87 or uPAR), plasminogen activator inhibitor and coagulation factor II (thrombin) receptor (Cf2r). In addition, by quantitative RT-PCR, 32D TPO clones expressed levels of Gata1 similar to those expressed by freshly isolated Mks (DeltaCt approximately 4.7 in both cases). In conclusion, the 32D TPO subclones described here are among the few pure Mk cell lines isolated so far and, for their unique properties, may prove themselves as a useful model to study Mk differentiation.
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http://dx.doi.org/10.1016/j.bcmd.2005.06.011DOI Listing
January 2006