In Vitro Erythroid Differentiation and Lentiviral Knockdown in Human CD34+ Cells from Umbilical Cord Blood
Human umbilical cord blood is a rich source of hematopoietic stem and progenitor cells. CD34+ cells in umbilical cord blood are more primitive than those in peripheral blood or bone marrow, and can proliferate at a high rate and differentiate into multiple cell types. In this protocol, a dependable method is described for the isolation of fetal CD34+ cells from umbilical cord blood and expanding these cells in culture. The cells can then be in vitro differentiated along an erythroid pathway, while simultaneously performing knockdown of a gene of choice. The use of lentiviral vectors that express small hairpin RNA (shRNA) is an efficient method to downregulate genes. Flow cytometric analyses are used to enrich for erythroid cells. Using these methods, one can generate in vitro differentiated cells to use for quantitative reverse transcriptase PCR and other purposes.
Key wordsUmbilical cord blood CD34+ cells Lentiviral infection shRNA knockdown Erythroid differentiation
We would like to thank the St. Louis Cord Blood Bank (SLCBB, St. Louis, MO) for providing us with fresh umbilical cord blood.
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