title: Serum-free generation of multipotent mesoderm (Kdr+) progenitor cells in mouse embryonic stem cells for functional genomics screening.
abstract: This unit describes a robust protocol for producing multipotent Kdr-expressing mesoderm progenitor cells in serum-free conditions, and for functional genomics screening using these cells. Kdr-positive cells are able to differentiate into a wide array of mesodermal derivatives, including vascular endothelial cells, cardiomyocytes, hematopoietic progenitors, and smooth muscle cells. The efficient generation of such progenitor cells is of particular interest because it permits subsequent steps in cardiovascular development to be analyzed in detail, including deciphering the mechanisms that direct differentiation. In addition, the oligonucleotide transfection protocol used to functionally screen siRNA and miRNA libraries is a powerful tool to reveal networks of genes, signaling proteins, and miRNAs that control the diversification of cardiovascular lineages from multipotent progenitors. Technical limitations, troubleshooting, and potential applications of these methods are discussed.
authors.affiliation: Sanford Burnham Medical Research Institute, La Jolla, California, USA.
authors.forename: Wesley L
authors.initials: WL
authors.lastname: McKeithan
authors.forename: Alexandre R
authors.initials: AR
authors.lastname: Colas
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Bushway
authors.forename: Saugata
authors.initials: S
authors.lastname: Ray
authors.forename: Mark
authors.initials: M
authors.lastname: Mercola
mesh_terms: D000818:Animals; D002021:Buffers; D018929:Cell Culture Techniques; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology*; D005819:Genetic Markers; D005821:Genetic Techniques; D023281:Genomics; D051542:Hepatocyte Nuclear Factor 3-beta / Q000378:metabolism; D057026:Induced Pluripotent Stem Cells / Q000166:cytology; D008648:Mesoderm / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D034741:RNA, Small Interfering / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
chemical_list: D002021:Buffers; D016895:Culture Media, Serum-Free; C496503:Foxa2 protein, mouse; D005819:Genetic Markers; D034741:RNA, Small Interfering; D051542:Hepatocyte Nuclear Factor 3-beta; D040301:Vascular Endothelial Growth Factor Receptor-2