Name: lentiviral-mediated knockdown during ex vivo erythropoiesis of human hematopoietic stem cells
Uploaded: 2011-07-16T14:00:00
Description: Watch this Scientific Journal Video about Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells at JoVE.com

हम पूर्व vivo erythroid भेदभाव, डी. एलन और एच. Atkins (Ohri, कनाडा) पर रक्त के नमूनों (ओटावा अस्पताल रिसर्च एथिक्स बोर्ड के तहत प्राप्त प्रदान करने के लिए सलाह के लिए एल Douay और एम सी Giarratana (Université पेरिस छठी, फ्रांस) धन्यवाद # ) 2007804-01H, डी. (इकोले पॉलीटेक्निक Federale डी लॉज़ेन) Trono गंभीर पांडुलिपि पढ़ने के लिए lentiviral वैक्टर और FJ Dilworth (Ohri, कनाडा) प्रदान करने के लिए. यह परियोजना एक CIHR अनुदान (एमओपी 82,813) के द्वारा MBCGP वित्त पोषित किया गया था एक ओंटारियो रिसर्च फंड कम्प्यूटेशनल Regulomics प्रशिक्षण Postdoctoral फैलोशिप के एक प्राप्तकर्ता है. MB जीन अभिव्यक्ति के नियमन में कनाडा अनुसंधान चेयर रखती है.

Part I. ex vivo erythropoiesis of human hematopoietic stem/progenitor cells
1. Isolation of CD34+ human hematopoietic stem/progenitor cells
Human CD34+ cell population, which contains a mixture of hematopoietic stem cells (HSCs) and early progenitors 8, 
is harvested from umbilical cord blood, peripheral blood mobilized with G-CSF (leukapheresis) or bone marrow (Figure 1-Step1). If using cord blood or peripheral 
blood, go direct...

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तरीकों की एक संख्या पहले से इस्तेमाल किया गया है संस्कृति में सफलता की चर डिग्री के साथ erythroid कोशिकाओं को अलग. उदाहरण के लिए, MS-5 पर सह संस्कृति के बिना कुछ प्रोटोकॉल erythroid कोशिकाओं के विस्तार की अनुमति देता ?...

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

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

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

एक<em&gt; पूर्व vivo</em&gt; प्रोटोकॉल परिपक्व मानव hematopoietic स्टेम / progenitors से लाल रक्त कोशिकाओं को उत्पन्न करने के लिए है वर्णित है. इसके अतिरिक्त हम प्राथमिक erythroid कोशिकाओं में प्रतिलेखन कारक TAL1 पछाड़ना एक कुशल lentiviral वितरण विधि का वर्णन. lentivirus मध्यस्थता जीन डिलीवरी की दक्षता GFP व्यक्त वायरस का उपयोग करते हुए प्रदर्शन किया है.

Lentiviral - मध्यस्थता नॉकडाउन के दौरान पूर्व vivo</emमानव hematopoietic स्टेम सेल के> erythropoiesis

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

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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

अलगाव और hematopoietic स्टेम सेल का प्रत्यारोपण (HSCs)

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

Murine भ्रूण स्टेम कोशिकाओं से hematopoietic स्टेम सेल की व्युत्पत्ति

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 ...

Passaging Human Neural Stem Cells

मानव तंत्रिका स्टेम कोशिकाओं passaging

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

अलगाव और अपरा से hematopoietic स्टेम सेल का विश्लेषण

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

प्रारंभिक murine जर्दी Sac और एजीएम से hematopoietic स्टेम सेल का अलगाव

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

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

भ्रूण स्टेम कोशिकाओं के Oligodendrocyte व्यापारियों में भेदभाव

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

पूर्व प्राथमिक मानव फैलोपियन ट्यूब उपकला कोशिकाओं के Vivo संस्कृति

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

प्ररूपी और murine hematopoietic स्टेम सेल और वंश प्रतिबद्ध progenitors के विश्लेषण से अलगाव

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

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

माउस भ्रूणीय त्वचा और Melanoblast प्रवासन के रहते इमेजिंग के पूर्व vivo संस्कृति

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

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

Confocal और Multiphoton माइक्रोस्कोपी का उपयोग पांच फ्लोरोसेंट प्रोटीन द्वारा चिह्नित hematopoietic स्टेम और पूर्वपुस्र्ष कोशिकाओं की विवो Clonal ट्रैकिंग में

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

Lentiviral - मध्यस्थता नॉकडाउन के दौरान पूर्व vivo erythropoiesis