Lentiviral - मध्यस्थता नॉकडाउन के दौरान पूर्व vivo erythropoiesis

Summary

एक<em> पूर्व vivo</em> 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.

Abstract

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.

Protocol

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…

Discussion

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 ?…