Wir danken L. Douay und MC Giarratana (Université Paris VI, Frankreich) für Beratung bei der ex vivo erythroiden Differenzierung, D. Allan und H. Atkins (Ohří, Kanada) für die Bereitstellung von Blutproben (erhalten unter der Ottawa Hospital Research Ethics Board # 2007804-01H), D. Trono (Ecole Polytechnique Fédérale de Lausanne) für die Bereitstellung der lentiviralen Vektoren und FJ Dilworth (Ohří, Kanada) für die kritische Durchsicht des Manuskripts. Dieses Projekt wurde von einem CIHR gewähren (MOP-82813), um MBCGP finanziert ist ein Empfänger einer Ontario Research Fund Computational Regulomics Ausbildung Postdoktoranden-Stipendium. MB hält den Canada Research Chair in der Regulation der Genexpression.

<ol>
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C., De Harven, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocytic+maturation+in+vitro+of+murine+(Friend)+virus-induced+leukemic+cells.">Erythrocytic maturation in vitro of murine (Friend) virus-induced leukemic cells.</a> Natl Cancer Inst Monogr. 22, 505-522 (1966).</li><li>Weiss, M. J., Yu, C., Orkin, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythroid-cell-specific+properties+of+transcription+factor+GATA-1+revealed+by+phenotypic+rescue+of+a+gene-targeted+cell+line.">Erythroid-cell-specific properties of transcription factor GATA-1 revealed by phenotypic rescue of a gene-targeted cell line.</a> Mol Cell Biol. 17, 1642-1651 (1997).</li><li>Lozzio, B. B., Lozzio, C. B., Bamberger, E. G., Feliu, A. 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Chapter 4, (2006).</li><li>Chao, M. P., Seita, J., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+normal+hematopoietic+and+leukemia+stem+cell+hierarchy.">Establishment of a normal hematopoietic and leukemia stem cell hierarchy.</a> Cold Spring Harb Symp Quant Biol. 73, 439-449 (2008).</li><li>Amsellem, S., Ravet, E., Fichelson, S., Pflumio, F., Dubart-Kupperschmitt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximal+lentivirus-mediated+gene+transfer+and+sustained+transgene+expression+in+human+hematopoietic+primitive+cells+and+their+progeny.">Maximal lentivirus-mediated gene transfer and sustained transgene expression in human hematopoietic primitive cells and their progeny.</a> Mol Ther. 6, 673-677 (2002).</li><li>Laurenti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+gene+and+shRNA+expression+in+resident+hematopoietic+stem+cells+in+vivo.">Inducible gene and shRNA expression in resident hematopoietic stem cells in vivo.</a> Stem Cells. 28, 1390-1398 (2010).</li><li>Palii, C. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+maturation+of+human+erythroid+progenitors+in+liquid+culture.">Proliferation and maturation of human erythroid progenitors in liquid culture.</a> Blood. 73, 100-103 (1989).</li><li>Wada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+major+blood+group+antigens+on+human+erythroid+cells+in+a+two+phase+liquid+culture+system.">Expression of major blood group antigens on human erythroid cells in a two phase liquid culture system.</a> Blood. 75, 505-511 (1990).</li><li>Sui, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythropoietin-independent+erythrocyte+production:+signals+through+gp130+and+c-kit+dramatically+promote+erythropoiesis+from+human+CD34++cells.">Erythropoietin-independent erythrocyte production: signals through gp130 and c-kit dramatically promote erythropoiesis from human CD34+ cells.</a> J Exp Med. 183, 837-845 (1996).</li><li>Panzenbock, B., Bartunek, P., Mapara, M. Y., Zenke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+of+human+stem+cell+factor/erythropoietin-dependent+erythroid+progenitor+cells+in+vitro.">Growth and differentiation of human stem cell factor/erythropoietin-dependent erythroid progenitor cells in vitro.</a> Blood. 92, 3658-3668 (1998).</li><li>Lindern, M. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glucocorticoid+receptor+cooperates+with+the+erythropoietin+receptor+and+c-Kit+to+enhance+and+sustain+proliferation+of+erythroid+progenitors+in+vitro.">The glucocorticoid receptor cooperates with the erythropoietin receptor and c-Kit to enhance and sustain proliferation of erythroid progenitors in vitro.</a> Blood. 94, 550-559 (1999).</li><li>Freyssinier, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=amplification+and+characterization+of+a+population+of+human+erythroid+progenitors.">amplification and characterization of a population of human erythroid progenitors.</a> Br J Haematol. 106, 912-922 (1999).</li><li>Malik, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+model+of+human+red+blood+cell+production+from+hematopoietic+progenitor+cells.">An in vitro model of human red blood cell production from hematopoietic progenitor cells.</a> Blood. 91, 2664-2671 (1998).</li><li>Carlile, G. W., Smith, D. H., Wiedmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-3+has+a+nonapoptotic+function+in+erythroid+maturation.">Caspase-3 has a nonapoptotic function in erythroid maturation.</a> Blood. 103, 4310-4316 (2004).</li><li>Mazurier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+and+efficient+selection+of+human+transduced+primitive+hematopoietic+cells+using+the+humanized+S65T+green+fluorescent+protein.">Rapid analysis and efficient selection of human transduced primitive hematopoietic cells using the humanized S65T green fluorescent protein.</a> Gene Ther. 5, 556-562 (1998).</li><li>Salmon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-level+transgene+expression+in+human+hematopoietic+progenitors+and+differentiated+blood+lineages+after+transduction+with+improved+lentiviral+vectors.">High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors.</a> Blood. 96, 3392-3398 (2000).</li></ol>

Eine Reihe von Methoden wurden bereits zu erythroiden Zellen in Kultur zu differenzieren mit variabler Grad von Erfolg eingesetzt. Zum Beispiel können einige Protokolle ohne Co-Kultur auf MS-5 Ausbau der erythroiden Zellen sind aber nicht in der Herstellung von voll ausgereiften entkernte rote Blutkörperchen 12-17 effizient. Während bei anderen Methoden effizienter Enukleation ermöglichen, ist es auf Kosten der Verbreitung 18,19. Die Methode, die wir hier verwendet haben, zunächst von der Doua...

lentiviral-mediated knockdown during ex vivo erythropoiesis of human hematopoietic stem cells

Erythropoese ist eine häufig verwendete Modellsystem zur Untersuchung der Zelldifferenzierung. Während Erythropoese, pluripotenten adulten humanen hämatopoetischen Stammzellen (HSZ) in oligopotent Vorfahren, engagiert Vorläufern und reifen roten Blutkörperchen Zellen 1 zu unterscheiden. Dieser Prozess ist für einen großen Teil auf der Ebene der Genexpression, wobei spezifische Transkriptionsfaktoren aktivieren Linie-spezifischer Gene reguliert, während gleichzeitig unterdrücken Genen, die spezifisch für andere Zelltypen 2 sind. Studien über Transkriptionsfaktoren regulieren Erythropoese sind oft unter Verwendung von humanen und murinen Zelllinien, stellen zum Teil erythroiden Zellen zu bestimmten Stadien der Differenzierung 3-5. Allerdings transformierten Zelllinien nur teilweise imitieren erythroiden Zellen und vor allem sie nicht erlauben es, nachvollziehbar Studie der dynamischen Veränderungen, die als Zellen des Fortschritts ist möglich durch viele Stadien zu ihrer endgültigen erythroiden Schicksal. Somit bleibt eine aktuelle Herausforderung der Entwicklung eines Protokolls zu relativ homogenen Populationen von primären HSC und erythroiden Zellen in verschiedenen Stadien der Differenzierung zu erhalten, die in Mengen, die ausreichen, Genomik und Proteomik Experimente durchzuführen sind. Hier beschreiben wir eine Ex-vivo-Zellkultur-Protokoll zu erythroiden Differenzierung von humanen hämatopoetischen Stammzellen / Vorläuferzellen, die aus entweder Nabelschnurblut, Knochenmark oder dem peripheren Blut Erwachsener mit G-CSF (Leukapherese) mobilisiert wurden, isoliert zu induzieren. Diese Kultur zeichnet sich anfänglich durch die Douay Labor 6 entwickelt, nutzt Zytokine und Co-Kultur auf mesenchymale Zellen in die Knochenmark-Mikroumgebung nachahmen. Mit dieser ex vivo Differenzierung Protokoll, beobachten wir eine starke Verstärkung von erythroiden Vorläuferzellen, eine Induktion der Differenzierung ausschließlich auf die erythroiden Linie und eine vollständige Reifung auf der Bühne des entkernten roten Blutkörperchen. So bietet dieses System die Möglichkeit, den molekularen Mechanismus der Regulation der Transkription als hämatopoetische Stammzellen Fortschritte auf dem erythroiden Linie zu studieren. Studieren Erythropoese auf der Transkriptionsebene erfordert auch die Fähigkeit, über-express oder Knockdown spezifische Faktoren in primären erythroiden Zellen. Zu diesem Zweck verwenden wir ein Lentivirus-vermittelte Gentransfer-System, das für die effiziente Infektion von beiden Teilen und nicht teilende Zellen 7 ermöglicht. Hier zeigen wir, dass wir in der Lage, effizient Knockdown des Transkriptionsfaktors TAL1 in primären humanen erythroiden Zellen sind. Darüber hinaus zeigt die GFP-Expression mit einem Wirkungsgrad von lentiviralen Infektion fast 90%. So bietet unser Protokoll ein sehr nützliches System für die Charakterisierung des regulatorischen Netzwerkes von Transkriptionsfaktoren, die Steuerung der Erythropoese.

Watch this Scientific Journal Video about Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells at JoVE.com

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Ein<em&gt; Ex vivo</em&gt; Protokoll zur Erzeugung reifen menschlichen roten Blutkörperchen aus hämatopoetischen Stammzellen / Vorläuferzellen beschrieben. Zusätzlich beschreiben wir eine effiziente lentivirale-Delivery-Methode Knockdown des Transkriptionsfaktors TAL1 in primären erythroiden Zellen. Die Effizienz der Lentivirus vermittelten Gentransfer nachgewiesen wird, mit GFP-exprimierenden Viren.

Lentivirale-vermittelte Knockdown Während Ex Vivo Erythropoese von humanen hämatopoetischen Stammzellen

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

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Research

JoVE Journal

Biology

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

13.0K Aufrufe.  Ottawa Hospital Research Institute. Ein Ex vivo Protokoll zur Erzeugung reifen menschlichen roten Blutkörperchen aus hämatopoetischen Stammzellen / Vorläuferzellen beschrieben. Zusätzlich beschreiben wir eine effiziente lentivirale-Delivery-Methode Knockdown des Transkriptionsfaktors TAL1 in primären erythroiden Zellen. Die Effizienz der Lentivirus vermittelten Gentransfer nachgewiesen wird, mit GFP-exprimierenden Viren.

Serie: Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Isolierung und Transplantation von hämatopoetischen Stammzellen (HSZ)

Forschung

Biologie

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Ableitung von hämatopoetischen Stammzellen aus embryonalen Stammzellen der Maus

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

In vitro und in vivo Biolumineszenz Reporter Gene Imaging von humanen embryonalen Stammzellen

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Isolierung und Analyse von hämatopoetischen Stammzellen aus der Plazenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

Isolierung von Anfang hämatopoetischen Stammzellen aus Murine Dottersack und AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Ex vivo-Kultur von primären humanen Eileiter Epithelzellen

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Phänotypische Analyse und Isolierung von murinen hämatopoetischen Stammzellen und Lineage-festgelegte Vorläuferzellen

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Ex-vivo-Kultur von embryonalen Maus-Haut-und-Live-Darstellung von Migration Melanoblasten

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

In vivo Klonale Verfolgung von hämatopoetischen Stamm-und Vorläuferzellen durch Fünf Fluoreszierende Proteine ​​mit Hilfe der konfokalen und Multiphotonen Mikroskopie Markierte

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In-vivo-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...